diarrhoea, dracunculiasis, hepatitis, typhoid fever and filariasis. ...... A septic tank is a watertight chamber where wastewater, both black water from the toilet and.
BACTERIOLOGICAL AND PHYSICOCHEMICAL QUALITIES OF WATER AND FISH SAMPLES FROM RIVER LAVUN, BIDA, NIGER STATE, NIGERIA
BY
Abdulmalik ALIYU B. Pharm. (A.B.U Zaria) 2002 P15PHPM8018
A DISSERTATION SUBMITTED TO THE SCHOOL OF POST GRADUATE STUDIES, AHMADU BELLO UNIVERSITY, ZARIA IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF SCIENCE DEGREE IN PHARMACEUTICAL MICROBIOLOGY.
DEPARTMENT OF PHARMACEUTICS AND PHARMACEUTICAL MICROBIOLOGY FACULTY OF PHARMACEUTICAL SCIENCES AHMADU BELLO UNIVERSITY, ZARIA NIGERIA
APRIL, 2016
DECLARATION
I declare that the work presented in this dissertation entitled “Bacteriological and Physicochemical Qualities of water and fish samples from River Lavun, Bida, Niger State, Nigeria” has been carried out by me in the Department of Pharmaceutics and Pharmaceutical Microbiology, under the supervision of Prof. Y. K. E. Ibrahim and Prof. (Mrs) R. A. Oyi. The information derived from literature has been duly acknowledged in the text and a list of references provided. No part of this thesis was previously presented for another degree or diploma at this or any other institution.
Abdulmalik ALIYU
__________________ Signature
i
____________ Date
CERTIFICATION
This dissertation entitled “BACTERIOLOGICAL AND PHYSICOCHEMICAL QUALITIES OF WATER AND FISH SAMPLES FROM RIVER LAVUN, BIDA, NIGER STATE, NIGERIA” by Abdulmalik Aliyu, meets the regulations governing the award of the degree of Master of Science of the Ahmadu Bello University, Zaria and is approved for its scientific contribution to knowledge and literary presentation.
Prof. Y. K. E. Ibrahim __________________________ Chairman, Supervisory Committee Signature Department of Pharmaceutics and Pharmaceutical Microbiology, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria.
_____________ Date
Prof. (Mrs) R. A. Oyi __________________________ Member, Supervisory Committee Signature Department of Pharmaceutics and Pharmaceutical Microbiology, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria.
_____________ Date
Dr. B.O. Olayinka Head of Department Department of Pharmaceutics and Pharmaceutical Microbiology, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria.
__________________________ Signature
_____________ Date
Prof. K. Bala Dean School of Post Graduate Studies Ahmadu Bello University, Zaria.
__________________________ Signature
_____________ Date
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ACKNOWLEDGEMENTS
I will like to express my profound gratitude and appreciation to Almighty Allah for His guidance, protection and sustenance coupled with good health through the course and successful completion of this program. I will ever live to remember and appreciate the unprecedented input and effort of my able supervisors, Prof. Y. K. E. Ibrahim and Prof. (Mrs) R. A. Oyi who have always been there for me. It is their dedication of time and energy for candid advice, thorough scrutiny, constructive criticism and supervisions that has put this work in the best shape. They will ever live to be remembered and reckoned in high esteem in my heart. I also want to appreciate and acknowledge my late father, Mal. Muhammad Abdulmalik Babatako and my beloved mother, Mallama Ayishat Mama Mahmud whose motherly care, concern, prayers and encouragement has brought me this far. I must appreciate the support, encouragement and prayers of my loving wife, Ummikulthum Abdulkadir Abdulmalik and our children: Mahmud, Amina and Abdulkadir. I also wish to express my sincere appreciation to my professors and able lecturers, Prof. J.A. Onaolapo, Prof. J.O. Ehinmidu, Prof. A.B. Isah, Prof. Hassan, Prof. (Mrs) G.O. Adeshina, Dr. B.O. Olayinka, Dr. B.A. Tytler, Dr. K. Mshelbwala, Dr. A.K. Olowosulu, Mr A. Falaki, Mr D. Ezekiel, Madam Victoria, Mr Godwin, Mr Bulus, Department of Fishery, Federal University of Technology (FUT), Minna, Mr J. Aliyu, Department of Microbiology, FUT, Minna and Mr Y.Y. Pala, Molecular Diagnostic Laboratory of Veterinary Teaching Hospital, ABU, Zaria. I must appreciate the contribution of my brothers and sisters in one way or the other to see to the successful completion of this work. They are Mal. Aliyu Ndatsonga and family, Mal. iii
Alhassan Ndaman and family, Mal. Alhassan Muhammad Baba and family, Mal. A. K. Mahamud, Mal. Ahmed Ndalamin, Mal. A. Ndayahaya, Mal. Umar A. Ibrahim, Mal. Umar A. Muhammad, Miss Umar Aisha and Miss Fatima Shuaib. I also appreciate the support of my in-laws particularly Hajiya Hadiza Abdulkadir, Abdullahi Nafada, Mal. Muhammad Abdulkadir Yacollege and many others. May Allah reward you all. This write up cannot be brought to conclusion without acknowledging the presence of my friends: Mr. I. James, Mr. N. Michael, Mr. D. Paul, Mal. Jiyah Dadi, Mal. A. Bishir, Mal. A. Abdulmuminni, Mal. M. Kachalla, Mal. A. Abdulfatai, Mal. M. Raji, Mal. A. Jibril, Mal. M. Musa Kudu, Mal. M. Mustapha, Mal. M. Kasim, Mal. A. Idris. etc. whose assistance and contributions I appreciated. What shall I say more, for the time will fail me to mention categorically individuals both home and abroad who have in one way or the other contributed to the success of this work, may God bless you all. Amen.
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DEDICATION
This work has been destined by Almighty Allah, and is dedicated to my ever supportive wife, Ummikulthum Abdulkadir Abdulmalik and our children.
v
Table of Contents DECLARATION ...................................................................................................................................... i CERTIFICATION .....................................................................................................................................ii ACKNOWLEDGEMENTS ....................................................................................................................... iii DEDICATION .........................................................................................................................................v LIST OF FIGURES ................................................................................................................................... x LIST OF TABLES .................................................................................................................................... xi LIST OF PLATE ..................................................................................................................................... xii LIST OF APPENDICES.......................................................................................................................... xiii ABBREVIATIONS ................................................................................................................................ xiv ABSTRACT ........................................................................................................................................... xv CHAPTER ONE ..................................................................................................................................... 1 1.1
INTRODUCTION ................................................................................................................... 1
1.2
Background of the Study ..................................................................................................... 1
1.3
Statement of Research Problem ......................................................................................... 5
1.4
Justification ......................................................................................................................... 8
1.5
Aim of the Study.................................................................................................................. 9
1.6
Specific Objectives .............................................................................................................. 9
1.7
Research Hypothesis ......................................................................................................... 10
1.8
Limitations and Challenges of the Study ........................................................................... 10
CHAPTER TWO................................................................................................................................... 11 2.0
LITERATURE REVIEW ......................................................................................................... 11
2.1
River Water ....................................................................................................................... 11
2.2
Water Pollution ................................................................................................................. 11
2.2.1
Sources of Water Pollution............................................................................................... 12
2.3
Types of Water Pollutants ................................................................................................. 15
2.3.1
Organic (Biologic) Pollutant .............................................................................................. 15
2.3.2
Physical and Chemical Pollutants ..................................................................................... 16
2.4
Water Pollution and Quality of Fish .................................................................................. 26
2.5
Prevention of Water Pollution .......................................................................................... 27
2.5.1 Source Water Protection ................................................................................................... 29 vi
2.5.2
Industrial Point-Source Pollution...................................................................................... 29
2.5.3
Agricultural Non-Point Source Pollution .......................................................................... 30
2.5.4
Settlements ...................................................................................................................... 31
2.6
Treatment of Water Pollution ........................................................................................... 32
2.6.1
Drinking Water Treatment ............................................................................................... 32
2.6.2
Treatment for Other Uses ................................................................................................ 34
2.7
Wastewater Treatment ..................................................................................................... 35
2.7.1 Domestic Wastewater Treatment ..................................................................................... 36 2.7.2
Industrial Wastewater Treatment .................................................................................... 39
2.7.3 Agricultural Wastewater Treatment ................................................................................. 40 2.8
Waterborne Diseases ........................................................................................................ 40
2.8.1
Typhoid Fever ................................................................................................................... 41
2.8.2
Dysentery.......................................................................................................................... 41
2.8.3
Cholera ............................................................................................................................. 42
2.8.4
Viral Gastroenteritis ......................................................................................................... 43
2.8.5
Prevention of Waterborne Diseases................................................................................ 43
CHAPTER THREE ................................................................................................................................ 45 3.0
MATERIALS AND METHODS .............................................................................................. 45
3.1
Study Area......................................................................................................................... 45
3.2.
Materials ........................................................................................................................... 47
3.2.1
Test Samples ..................................................................................................................... 47
3.2.2
Reagents and Diagnostics ................................................................................................. 47
3.2.3
Culture Media ................................................................................................................... 47
3.2.4
Equipment ........................................................................................................................ 48
3.2.5
Antibiotic Discs ................................................................................................................. 48
3.3
Methods ............................................................................................................................ 49
3.3.1 Collection of water samples .............................................................................................. 49 3.3.2
Preparation of Water Samples for Elemental Analysis .................................................... 49
3.3.3
Collection of Fish Samples ................................................................................................ 50
3.3.4
Preparation of Fish Samples for Microbiological Analysis................................................ 50
3.3.5
Preparation of Fish Samples for Elemental Analysis ........................................................ 50
3.3.6
Preparation of Bacteriological Media ............................................................................... 51 vii
3.3.7
Heterotrophic (Standard) Plate Count ............................................................................. 51
3.3.8
Determination of Faecal Coliform Count ......................................................................... 51
3.3.9
Faecal Streptococci Count ................................................................................................ 52
3.3.10 Preliminary Identification of the Isolates ........................................................................ 52 3.3.11 Biochemical Tests ............................................................................................................. 53 3.3.12 Identification of the Oxidase-Negative Enterobacteriaceae Family ................................ 54 3.3.13 Preparation of Barium Sulphate Standard (McFarland 0.5)............................................. 56 3.3.14 Standardization of Innocula ............................................................................................. 56 3.3.15 Antibiotic Susceptibility Testing ....................................................................................... 57 3.3.16 Determination of Multiple Antibiotic Resistance (MAR) Index........................................ 57 3.3.17 Plasmid Curing .................................................................................................................. 58 3.3.18 Molecular Characterization of some Antibiotic-Resistant Isolates ................................. 58 3.3.19 Physicochemical Analysis of the Water Samples ............................................................ 61 3.3.20 Determination of Elemental Composition of Water and Fish Samples .......................... 65 CHAPTER FOUR ................................................................................................................................. 66 4.0
RESULTS............................................................................................................................. 66
4.1
Bacteriological Analysis of River Lavun Water Samples.................................................... 66
4.2
Bacteriological Analysis of Fish ......................................................................................... 66
4.3
Distribution of Isolates in Water and Fish Samples .......................................................... 71
4.4
Antibiotic Susceptibility Profiles of the Isolates ................................................................ 72
4.5
Determination of MAR Index ............................................................................................ 80
4.6 Resistance Pattern of Some Antibiotic Resistant Bacteria Species Isolated From River Water And Fish Caught From River Lavun. ................................................................................... 80 4.7
Plasmid Analysis ................................................................................................................ 84
4.8
Physicochemical Properties of Water Samples ................................................................. 84
4.9
Elemental Analysis of Water and Fish Samples ................................................................ 91
CHAPTER FIVE.................................................................................................................................... 96 5.0
DISCUSSION ....................................................................................................................... 96
CHAPTER SIX .................................................................................................................................... 106 6.0
SUMMARY, CONCLUSION AND RECOMMENDATION ..................................................... 106
6.1
Summary ......................................................................................................................... 106
6.2
Conclusion ....................................................................................................................... 108 viii
6.3
Recommendation ............................................................................................................ 109
REFERENCES .................................................................................................................................... 110 APPENDICES .................................................................................................................................... 120
ix
LIST OF FIGURES
Fig. 3.1: Map of study area showing sampling points on River Lavun
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46
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67
Fig. 4.1: The mean values of HPC, FCC and FSC in April, 2014 (Early rainy season) -
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Fig. 4.2: The mean values of HPC, FCC and FSC in September, 2014 (Peak of rainy season)
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68
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69
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70
Fig. 4.5: Composition of Enterobacteriaceae contaminants in water samples
74
Fig. 4.6: Composition of Enterobacteriaceae contaminants in fish samples
75
Fig. 4.3: Comparison of HPC, FCC and FSC at point C
Fig. 4.4: The mean values of HPC and FCC of fish from April to September, 2014
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Fig. 4.7: Composition of Staphylococcus species contaminants in water and fish samples -
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76
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88
Fig.4.8: The mean values of DO, BOD and COD from April to September, 2014 -
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Fig. 4.9: The mean values of pH, Conductance and Alkalinity from April to September, 2014
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89
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90
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93
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94
Fig. 4.10: The mean values of Nitrate and Phosphate from April to September, 2014
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Fig. 4.11: Concentrations of some elements in water samples at sampling Points in August, 2014
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Fig. 4.12: Concentration of some elements at sampling point C from April to August, 2014
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x
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LIST OF TABLES Table 4.1: Distribution of bacteria Isolates from water and fish samples Collected from River Lavun
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73
Table 4.2: Antibiotic Resistance Profiles of some Enterobacteriacea Bacteria Isolated from River Lavun -
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77
Table 4.3: Antibiotic Resistance Profiles of some Enterobacteriacea Bacteria Isolated from fish caught from River Lavun
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78
Table 4.4: Antibiotic Resistant Profiles of Staphylococcus spp. isolated from water and fish samples collected from River Lavun
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79
Table 4.5: MARI of bacteria isolates from water and fish samples collected From River Lavun -
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81
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Table 4.6: Resistant pattern of some Antibiotic Resistant bacteria species Isolates from water and fish samples collected from river Lavun before and after curing
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Table 4.7: Comparison of resistance of some antibiotics to twenty-two Multiple Antibiotic Resistant bacteria species isolated from water and fish Caught from River Lavun before and after plasmid curing
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83
Table 4.8: Comparison of Plasmid number and sizes of some antibiotic resistant bacteria isolated from water and fish caught from River Lavun -
86
Table 4.9: Concentration of elements in water collected from River Lavun at sampling points B and C-
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92
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Table 4.10: Concentration of elements in fish collected from River Lavun at point C from April to August, 2014
xi
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LIST OF PLATE Plate 4.1: Gel electrophoresis of 100 base pair and plasmid DNA isolated Multiple Drug Resistant Isolates from River Water and fish caught from River lavun -
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xii
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85
LIST OF APPENDICES Appendix I: River Water Heterotrophic (Standard) Plate Count (cfu/ml)
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120
Appendix II: River Water Faecal Coliform Count (cfu/ml) -
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120
Appendix III: River Water Faecal Streptococcal Count (cfu/ml)
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120
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121
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121
Appendix VI: EUCAST Break points, 2014 (Staphylococcus spp.) -
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122
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123
Appendix IV: Heterotrophic plate count (HPC) and Faecal coliform count (FCC) of fish caught from River LavunAppendix V: EUCAST Break points, 2014 (Enterobacteriaceae)
AppendixVI: Interpretation of Antibiotic Susceptibility Tests of the Different Bacterial Isolates
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Appendix VII: Resistance pattern of some Antibiotic Resistant Bacteria Species isolated from river water and fish caught from River Lavun -
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127
Appendix VIII: Physico-chemical Parameters of April, 2014
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128
Appendix IX: Physico-chemical Parameters of May, 2014 -
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128
Appendix X: Physico-chemical Parameters of June, 2014
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128
Appendix XI: Physico-chemical Parameters of July, 2014 -
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129
Appendix XII: Physico-chemical Parameters of August, 2014
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129
Appendix XII: Physico-chemical Parameters of September, 2014 -
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129
Appendix XIII: Guidelines on drinking water and fish
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131
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Appendix XIV: Concentration of elements in water collected from River Lavun at sampling Point A -
xiii
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ABBREVIATIONS AAS: Atomic Absorption Spectrophotometer AWWA: America Water Works Association DDT: Dichloro Diphenyl Trichloroethane DNA: Deoxyribose Nucleic Acid E.M.B: Eosine Methylene Blue EUCAST: European Committee on Antimicrobial Susceptibility Testing NIS: Nigeria Industrial Standard FAO: Food and Agricultural Organization MIC: Minimum Inhibitory Concentration UNEP: United Nations Environmental Protection USFDA: United States Food and Drug Administration UV: Ultra Violet WHO: World Health Organization
xiv
ABSTRACT River Lavun is an important water source in Bida axis of Niger State, Nigeria especially with regard to domestic use, irrigation and aquatic food. Water quality is often altered by anthropogenic activities, it is therefore, necessary to evaluate bacteriological and physicochemical quality of this water source and its fish to determine its impacts on the health of the populace in this locality. Bacterial counts of the water revealed high contamination, with highest heterotrophic plate, faecal coliform and faecal streptococci counts observed in September, peak of rainy season. However, microbial counts of fish revealed that both heterotrophic and faecal coliform counts were higher than that of river water. Forty-seven bacterial isolates belonging to different bacterial families and genera were identified in the water samples while in the fish samples, fifty-three isolates belonging to twenty different species were identified using Microbact GNB 12E and Microbact Staphylococcal 12S (Oxoid). Predominant bacteria isolated included Klebsiella spp. (31%), Staphylococci spp. (20%), Enterobacter spp. (17%) and Escherichia coli (12%). Other isolates included Salmonella spp. (7%), Serratia and Citrobacter spp. (5% each), Shigella spp. (2%) and Yersinia enterocolitica (1%). The Enterobacteriaceae constituted 80% of the isolates. Antibiogram carried out using disc diffusion technique showed multiple antibiotic resistances of Serratia spp., Klebsiella spp., Enterobacter spp., Escherichia coli, Salmonella spp. and Citrobacter spp. to erythromycin, tetracycline, ampicillin, amoxicillin-clavulanate and cefuroxime. In contrast, virtually all the enteric bacterial isolates showed marked susceptibilities to ciprofloxacin, nitrofurantoin and gentamicin. However, Staphylococcus xv
spp. were generally susceptible to virtually all the antibiotics tested. Multiple Antibiotic Resistant Index, MARI of 0.3 and above were exhibited by 82% of the isolates. Of the 19 high MARI (>0.5) isolates subjected to acridine orange treatment, 62-74% became susceptible to most of the antibiotics previously resistant to. Presence of plasmids were confirmed in 13 isolates with molecular weights in the range of 1.6kbp to 3.2kbp. The physicochemical parameters studied included temperature, pH, alkalinity, conductivity, phosphate, nitrate, biochemical oxygen demand (BOD) and chemical oxygen demand (COD) which were within acceptable limit set by World Health Organization (WHO) and/or Nigeria Industrial Standard (NIS)). The elemental analysis was carried out showed that Silver, Cobalt, and Lead, were not detected in the water. The results further revealed that Iron, Manganese, Zinc, Copper, Nickel, and Cadmium, concentrations were above acceptable limit of drinking water set by WHO and/or NIS while Iron, Zinc and Copper were above permissible limit in fish prescribed by FAO/WHO.
xvi
CHAPTER ONE 1.1
INTRODUCTION
1.2
Background of the Study
Water is essential to life, and a satisfactory (adequate, safe and accessible) supply must be available to all (WHO, 2011). The importance of water to man is aptly summarized in the words of Kofi Annan who said: “Access to safe water is a fundamental human need and, therefore, a basic human right. Contaminated water jeopardizes both the physical and social health of people. It is an affront to human dignity’’ (WHO, 2002). Water is vital to the existence of all living organisms, but this valued resource is increasingly threatened as human populations grow with increase demand for more water for domestic purposes and economic activities (UNEP, 2000). Human settlements are often dictated by the nearness to source of surface or ground waters. The quality of any body of water is a function of either natural influences or human activities or both (Stark et al., 2001; Kolawale et al., 2008). UNEP (2000) stated that aquatic environments cannot be perceived simply as holding tanks that supply water for human activities. Rather, these environments are complex matrices that require careful use to ensure sustainable ecosystem functioning well into the future. One of the most abundant and readily available source of surface water is River. It is the most important freshwater source for man. Unfortunately, river waters are polluted by indiscriminate disposal of sewage, industrial waste and plethora of human activities, which affect their physicochemical characteristics and microbiological qualities (Koshy et al., 1999). Pollution of the aquatic environment is a serious and growing problem. Increasing numbers and amounts of industrial, agricultural and commercial chemicals discharged into 1
the aquatic environment have led to deleterious effects on aquatic organisms. Aquatic organisms, including fish, accumulate pollutants directly from contaminated water and indirectly via the food chain (Hammer, 2004; Mohammed, 2009; Yogendra et al., 2013; Torimiro et al., 2014). Owing to the large quantity of effluents discharged into receiving waters, the natural processes of pathogen reduction are inadequate for protection of public health. In addition, industrial wastes that alter the water pH and provide excessive bacterial nutrients often compromise the ability of natural processes to inactivate and destroy pathogens (Gerardi et al., 2005). The extent of discharge of domestic and industrial effluents is such that rivers receiving untreated effluent cannot provide the dilution necessary for their survival as good quality water sources. Disposal of sewage wastes into a large volume of water could increase the biological oxygen demands (BOD) to such a high level that all the available oxygen may be removed, consequently causing the death of all aerobic species, including, fish (Maduka, 2004). One of the most important sources of water pollution is microbial contamination; especially with pathogenic microorganisms. Enteric pathogens are typically responsible for waterborne sickness (Raji and Ibrahim, 2011). Contamination of water is a serious environmental problem as it adversely affects human health and biodiversity in the aquatic ecosystem. The use of indicator bacteria such as faecal coliforms (FC) and faecal streptococci (FS) for assessment of faecal pollution and possible water quality deterioration in fresh water sources is generally recommended (APHA, 1995).
2
Currently, coliforms and E. coli are of great importance among bacterial indicators used in water quality and health risk definition (WHO, 2011). Pathogens are a serious concern for managers of water resources, because excessive amounts of faecal bacteria in sewage and urban run-offs have been known to indicate risk of pathogen-induced illnesses in humans (Adeyinka et al., 2014). Several species of Gram-negative bacteria present in municipal wastewater are pathogenic. Fawole (2007) reported that fish from rivers are now receiving increasing attention as potential source of animal protein and essential nutrients for human diets. Fish meat is known for its high nutritional quality, relative low fat content, saturated fat, cholesterol and high levels of poly unsaturated fatty acids, proteins and minerals such as calcium, phosphorous, sodium, potassium and magnesium (Salihu et al., 2012), hence are favoured over other white meats. In the past, it was thought that fish harvested from open waters (marine and fresh) were generally safe, principally because of the practice of quick chilling of fish and fisheries products soon after harvesting. This notion, according to Reilly et al. (1997) was borne out of the lack or paucity of epidemiological evidence of fish-borne diseases. Recent evidence from fisheries reports and studies in the areas of water pollution, fish handling and preservation, water management/fish feeding practices in aquaculture and some cultural practices of fish preparation and raw fish consumption have suggested otherwise (Reilly et al., 1997; Atiribom et al., 2007; Obasohan et al., 2010; Olayiwola and Adedokun, 2015). The expansion of fish production facilities in the effort to meet animal protein supply through increased fish production has placed increased requirements of quality and product safety on
3
producers, marketers and regulators. This assertion was emphasized by Ihuahi and Omojowo (2005), which opined that the issue of quality and safety of fish and fisheries products have become a serious concern to consumers and regulators in both producing and importing countries. Pathogenic microbes cause many diseases in both wild and cultured fish. They may vary from a primary pathogen to that of an opportunist invader of a host rendered moribund by some disease process (Inglis et al., 1994). Fish may harbour pathogens on or inside its body after exposure to contaminated water or food. Most commonly reported pathogens in fish include: Samonella, Shigella, Leptospira, E. coli, Vibrio, Mycobacterium spp., viruses and hookworm larvae. Salihu et al. (2012) reported the isolation of bacterial pathogens from fish caught from River Sokoto. Some of these bacterial isolates were E. coli, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus vulgaris and Salmonella typhimurium. It has been reported that human diseases that can be caused by bacteria in fish include:
Food poisoning and gastroenteritis by Samonella, Vibrio and Clostridium spp., and Campylobacter jejuni (Davis et al., 1967).
Diarrhoea by Edwardsiella sp., Staphylococus sp., Escherichia sp. and Aeromonas sp. (Davis et al., 1967; Inglis et al., 1994).
Superficial wound infections, and ulcers, due to Pseudomonas sp. (Obasohan et al., 2010).
Bacillary dysentery (Shigellosis) caused by Shigella sp. (Cheesbrough, 2006).
Clonorchiasis, Dracunculiasis and Paragonimiasis due to larvae and metacercariae ingested in fish and crustaceans (Cheesbrough, 2006).
4
Cholera caused by Vibrio cholerae (Atiribom et al., 2007).
Typhoid and paratyphoid fevers due to Samonella typhi and Samonella paratyphi respectively. (Nyaku et al., 2007).
1.3
Statement of Research Problem
An adequate supply of water in terms of quality and quantity is a primary requisite for good health. Provision of adequate and safe water for the two third of the world’s population is still a dream. In Nigeria, water-related diseases are among the five leading causes of death in children lower than five years of age (FAO, 1994). The most common and wide spread health risk associated with drinking water is microbial contamination. This could be caused directly or indirectly by human or animal excreta. If such contamination is recent and emanates from carriers of communicable diseases, drinking such contaminated water or using it in food preparation may result in infection. In nearly all epidemics of water borne diseases, it has been observed that the bacteriological quality of water was unsatisfactory (Galbraith, 1994). Raji and Ibrahim (2011) and Adeyinka et al. (2014) revealed that the common diseases of drinking water in Nigeria include cholera, diarrhoea, dracunculiasis, hepatitis, typhoid fever and filariasis. Olatunji et al. (2011) in a study on Asa River at Ilorin, Nigeria, reported that the alarming high number of total coliforms and thermotolerant (faecal) colifoms per 100 mL obtained from the water samples, which exceeds at least ten times the recommended limit, indicates high level of faecal pollution of the river water, and that this potentially poses a high health risk for recreational purposes, let alone for drinking purpose. This clearly implies that the organic pollution of the river is more of faecal pollution.
5
Oyeleke and Istifanus (2008) reported the isolation of pathogenic organism like E. coli, Streptococcus faecalis, Aeromonas hydrophilla, Shigella sonnei, Salmonella typhi and Chromobacterium violaceum from River Kaduna. These bacteria have been reported as causative agents of various diseases (Dennis et al., 2005). Similarly, Tytler (2011), in his study on River Kaduna showed that members of the Enterobacteriaceae were the most common organisms isolated (62.44%) with E. coli being most common followed distantly by S. aureus and Ps. aeroginosa. In Nigeria, fish is the preferred source of the much desired animal protein compared to poultry, beef, mutton, pork and veal. It is comparatively cheaper and highly acceptable, with little or no religious bias, which gives it an advantage over pork or beef (Feldhusen, 2000). Noga (2000) observed that the prevalence of infectious diseases depends on the interaction between the fish and the pathogens of the aquatic environment, hence the bacteria flora of the fish depicts the level of bacteria in the water environment (Torimiro et al., 2014). Salihu et al. (2012) also revealed the presence of Enterobacteriaceae and some Gram-positive bacteria in fish from Sokoto River. These contaminated fish if consumed could cause serious health problems to human population. Chemicals, organic and inorganic, from industrial waste, mining and agricultural activities could find way into lakes, rivers and streams through open drains and gutters as such the human activities deteriorates the quality of these water bodies with chemicals which cause an increase in heavy metal ion concentration in such water environment which could lead to elemental poisoning, when the water is consumed, if the concentration exceeds the acceptable limit. 6
Ozparlak and Arslan (2012) reported that metal pollutants in the form of particles, metal ions, and organic and inorganic compounds would make the aquatic environment toxic. Olagoke and Olatunji (2014) in their study on Rivers Lanzun and Tukwagi which flow through Bida town and drains from River Lavun, revealed that Cu, Zn, Fe, Mn and Cr were detected in concentrations above WHO limits. Elemental toxicants could enter fish directly through the digestive tract due to consumption of contaminated water and food or non-dietary routes across permeable membranes such as gills (Burger et al., 2002). Goldstein (1990) and Malik (2004) have revealed that fish acts as a bio-indicator of heavy metal levels in aquatic environment and when their concentrations exceed the required levels, they became toxic and cause several health problem. Therefore introduction of heavy metals into food chain threatens human health. Odoemelan (2005) reported an accumulation of heavy metals such as Ni, Cu, Mn, Pb, Zn, Fe, Hg, Cr, V and Cd in fish from Oguta Lake in Nigeria. Different fish samples from Kaduna River in Nigeria have been analyzed for toxic elemental contaminants such as Hg, Cd, V, Zn and Fe which were identified in appreciable amount in all the fish samples studied (Nwaedozie, 1998). Since fish have been recognized as good bio-accumulators of organic and inorganic pollutants (King and Jonathan, 2003) these contaminants cause unhealthy effects to the fish and this may be transferred to man through contaminated fish. River Lavun is the major source of drinking water in Bida and its environs in Niger State. The major occupation of the people around the river is farming. Run-off of fertilizers, pesticides, herbicides and other organic and inorganic materials from agricultural land eventually end up in the river.
7
Other human activities around the river involved fishing, washing, transportation, irrigation and mining. Fish from this River is not only consumed by the local populace it is also sold to people from various parts of the country. These human activities on and around the river would cause serious problems in the quality of the river water and its fish. In 2011, there was an outbreak of cholera in Lavun Local Government Area of Niger state which claimed lives (Abu, 2011). There is, at present no information on the microbiological and chemical quality of the water from this river, the fish caught from it, and the attendant health hazards. This study, therefore, sets out to address those challenges.
1.4
Justification
A sizeable proportion of the populace particularly in developing countries are highly prone to water-borne infections, majorly at the early period of the rainy season, with gastrointestinal infection like cholera, diarrhoea, and dysentery being predominant. River water in most developing countries serves as source of water to the rural communities. In most villages, there are inadequate infrastructure to treat the water so it is used directly (raw/untreated) and this has led to increase in water-borne diseases. River Lavun, which is the lower part of River Kaduna is the major source of water for the communities in Bida and its environs for their domestic uses (drinking, cooking and washing), fishing, farming and various other recreational activities. Fishing on the river by the people is a major economic activities in this environs. Microbiological contamination and chemical pollution of the River and its fish will have great impact on the health of the communities and other consumers of the fish from this river. 8
Data on the microbiological quality of fish from this river will provide information for proper measures for the prevention of disease outbreaks that may occur and institution of management and control measures.
1.5
Aim of the Study
The aim of this study is to determine the bacteriological and physicochemical qualities of water and fish samples from River Lavun with a view to controlling or minimizing infection sand untoward human health effects from this source.
1.6 i.
Specific Objectives Evaluation of the general bacteriological quality of River Lavun (heterotrophic, coliform and streptococci counts) and its fish (microbial level), and identification of isolates, etc. using standard microbiological techniques.
ii.
Determination of the antibiogram of the bacterial isolates, and characterization of the profiles of resistant isolates by the agar disc diffusion test and plasmid DNA analysis.
iii.
Assesment of the physicochemical quality of the water samples such as DO, BOD, COD, etc. using standard methods.
iv.
Examination of the elemental composition of the water and fish samples from the River using Atomic Absorption Spectrophotometer (AAS).
9
1.7
Research Hypothesis
The following hypotheses are formulated:
Null Hypothesis (Ho) 1 - River Lavun and do not contain any known pathogenic bacteria and hazardous chemicals.
Null Hypothesis (Ho) 2 - The fish from River Lavun are not associated with pathogenic organisms and hazardous chemicals.
Alternate Hypothesis (Hi) 1 - River Lavun is microbiologically unsafe and chemically polluted.
Alternate Hypothesis (Hi) 2 - The fish from River Lavun are associated with pathogenic organisms and chemically polluted.
1.8
Limitations and Challenges of the Study
This study specifically has, as its focus, the assessment of physico-chemical quality of the section of River Lavun at Wuya Kpata, Bida.
Sample collection of water and fish was carried out between the months of April and September, 2014.
Only one species of fish, Clarias gariepinus (Cat fish), was analysed in this study.
The direct impact of the chemical and bacteriological pollution on the health of the people living at Wuya Kpata and environs was not assessed.
A major constrain was flooding which occurred during the months of August and September. At this time, the sampling points were not accessible.
10
CHAPTER TWO
2.0
LITERATURE REVIEW
2.1
River Water
Rivers are the most important freshwater resource for man. Major river water uses include drinking, irrigation, industrial and municipal supply and waste disposal, navigation, fishing, boating, recreation and aesthetic value (Meybeck et al., 1996). Through these uses of river water, aquatic ecosystem is altered and the quality of river water is affected. Water is said to be of good quality if its chemical, biological and physical characteristics are within the acceptable limit set up for that purpose. In most cases, water quality refers to water for drinking, swimming, and fishing. When water is impaired by anthropogenic contaminants and make its unsafe for human, such as drinking and could not support its aquatic life, it is referred to as being polluted. Owa (2014) reported that human activities including industrialization and agricultural practices contributed immensely to the water quality which adversely has an effect on the water bodies (rivers and ocean) that is a necessity for life, that is why despite the fact that 70% of earth is covered with water, hardly 2% of it is drinkable (Udaybir et al. , 2014).
2.2
Water Pollution
According to WHO, one sixth of the world’s population, approximately 1.1 billion people do not have access to safe water and 2.4 billion lack basic sanitation (EPHA, 2009). Water pollution causes approximately 14,000 death per day, mostly due to contamination of drinking water by untreated sewage in developing countries (Owa, 2014). Addition or presence of undesirable substances in water is called water pollution. Tytler (2011) defined 11
water pollution as any biological, chemical, or physical change in water quality that has a harmful effect on living organisms or makes water unsuitable for desired users. Water pollution is one of the most serious environmental problems, is caused by a variety of human activities such as industrial, agricultural and domestic. Agricultural run-off laden with excess fertilizers and pesticides, industrial effluents with toxic substances and sewage water with human and animal wastes pollute our water thoroughly. Natural sources of pollution of water are soil erosion, leaching of minerals from rocks and of organic matter (Contemporary Environmental Issues, [C.E.I], 2015). Rivers, lakes, seas, oceans, estuaries and ground water sources may be polluted by point or non-point sources. When pollutants are discharged directly from a specific location such as a drain pipe carrying industrial effluents into a water body, it is said to be a point source pollution. In contrast, non-point sources include discharge of pollutants from diffused sources or from a larger area such as run off from agricultural fields, grazing lands, construction sites, abandoned mines and pits, roads and streets (C.E.I, 2015).
2.2.1 Sources of Water Pollution Water pollution is the major source of water borne diseases and other health problems. Sediments brought by runoff water from agricultural fields and discharge of untreated or partially treated sewage and industrial effluents, disposal of fly ash or solid waste into or close to a water body cause severe problems of water pollution. Increased turbidity of water because of sediments reduces penetration of light in water that reduces photosynthesis by aquatic plants (C.E.I, 2015). i.
Pollution due to pesticides and inorganic chemicals
12
Pesticides like dichloro dipheny trichloroethane (DDT), and other chemical substances used in agriculture may contaminate water bodies. Aquatic organisms take up pesticides from water get into the food chain (aquatic in this case) and move up the food chain. At higher trophic level, they get concentrated and may reach the upper end of the food chain (C.E.I, 2015).
Metals like lead, zinc, arsenic, copper, mercury and cadmium in industrial waste waters adversely affect humans and other animals. Arsenic pollution of ground water has been reported from West Bengal, Orissa, Bihar, Western U.P. Consumption of such arsenic polluted water leads to accumulation of arsenic in the body parts like blood, nails and hairs causing skin lesions, rough skin, dry and thickening of skin and ultimately skin cancer (C.E.I, 2015).
Oil pollution of sea occurs from leakage from ships, oil tankers, rigs and pipelines. Accidents of oil tankers spill large quantity of oil in seas which kills marine birds and adversely affects other marine life and beaches (C.E.I, 2015).
ii.
Thermal pollution Power plants- thermal and nuclear, chemical and other industries use a lot of water (about 30% of all abstracted water) for cooling purposes and the used hot water is discharged into rivers, streams or oceans. The waste heat from the boilers and heating processes increases the temperature of the cooling water. Discharge of this heat into water bodies, often referred to as thermal pollution. This heat may increase the temperature of the receiving water by 10 to 15 °C above the ambient water temperature. Increase in water temperature decreases dissolved oxygen in water which adversely affects aquatic life. Unlike terrestrial ecosystems, the temperature of water bodies remain steady and does not change very much. Accordingly,
13
aquatic organisms are adopted to a uniform steady temperature of environment and any fluctuation in water temperature severely affects aquatic plants and animals. Hence discharge of hot water from power plants adversely affects aquatic organisms. Discharge of hot water in water body affects feeding in fishes, increases their metabolism and affects their growth. Their swimming efficiency declines. Running away from predators or chasing prey becomes difficult. Their resistance to diseases and parasites decreases. Due to thermal pollution biological diversity is reduced. One of the best methods of reducing thermal pollution is to store the hot water in cooling ponds, allow the water to cool before releasing into any receiving water body (C.E.I, 2015). iii.
Ground water pollution
Lot of people around the world depend on ground water for drinking, domestic, industrial and agricultural uses. Generally groundwater is a clean source of water. However, human activities such as improper sewage disposal, dumping of farm yard manures and agricultural chemicals, industrial effluents are causing pollution of ground water (C.E.I, 2015). iv.
Eutrophication ‘Eu’ means well or healthy and ‘trophy’ means nutrition. The enrichment of water bodies with nutrients causes eutrophication of the water body. Discharge of domestic waste, agricultural surface runoff, land drainage and industrial effluents in a water body leads to rapid nutrients enrichment in a water body. The excessive nutrient enrichment in a water body encourages the growth of algae duckweed, water hyacinth, phytoplankton and other aquatic plants. The biological oxygen demand (BOD) increases with the increase in aquatic organisms. As more plants grow and die, the dead and decaying plants and organic matter
14
acted upon by heterotrophic protozoans and bacteria, deplete the water of dissolved oxygen (DO). Decrease in DO result in sudden death of large population of fish and other aquatic organisms including plants, releasing offensive smell and makes the water unfit for human use. The sudden and explosive growth of phytoplankton and algae impart green colour to the water is known as water bloom, or “algal blooms”. These phytoplankton release toxic substances in water that causes sudden death of large population of fishes. This phenomenon of nutrient enrichment of a water body is called eutrophication. Human activities are mainly responsible for the eutrophication of a growing number of lakes and water bodies in the country (C.E.I, 2015).
2.3
Types of Water Pollutants
Usually the cause of the pollution determines its type. The different types are organic (biologic), chemical (such as nutrients, pesticides and herbicides) and physical pollutions.
2.3.1 Organic (Biologic) Pollutant A number of water pollutants are organic in nature such as human salvage, animal waste, and plant residue. Though bacteria helps in decomposing the organic matter, human and animal waste can also contain pathogenic bacteria, viruses, or protozoa. Hogan (2010) reported that pathogen can produce water borne diseases in either human or animal hosts. Hunter (2003) reported the increase in cases of water-borne diseases in United States of America since 1940 with increase in number of individual affected per outbreak. WHO (2000) estimated that water-borne diseases could cause more than two million deaths and four billion cases of diarrhoea annually. In 2011, WHO reported that infectious diseases caused by pathogenic bacteria, viruses and parasites (e.g. protozoa and helminthes) are the most common and widespread health risk associated with drinking water. 15
Abednego et al. (2013) in its work on River Nairobi, revealed that microbiological quality of the surface water was unacceptably higher than compliance level of the National and the WHO guidelines for drinking water and agriculture use and that the pathogenic bacteria include Escherichia coli, Klebsiella aerogenes, shigella flexneri, Enterococcus faecalis, Salmonella paratyphi, Pseudomonas aeruginosa, and Vibrio cholerae. Some of these bacteria have been reported to cause water borne diseases in Nigeria such as cholera, typhoid fever, dysentery, diarrhea, dracunculiasis, hepatitis and filariesis (Raji and Ibrahim, 2011; Adeyinka et al., 2014). Noticeaably amongst the water borne disease bacteria is coliform bacteria which is commonly used as bacterial indicator of water pollution. The bacterial requirement for drinking water is zero per 100ml sample for total coliform, faecal coliform, and Escherichia coli (WHO, 2011). Faecal indicator bacteria like total coliforms, faecal coliforms (thermotolerant coliforms), E. coli and intestinal enterococci (faecal streptococci) are excreted by humans and warm blooded animals, pass sewage treatment plants to a great extent and survive for a certain time in the aquatic environment (Kavka and Poetsch, 2002).
2.3.2 Physical and Chemical Pollutants Chemical contaminants may include organic and inorganic substances. Organic water pollutants include detergents, disinfection by-products, and food processing wastes. Others are insecticides, herbicides, and petroleum hydrocarbons, while inorganic water pollutants are ammonia, chemical waste, fertilizers, heavy metals and silt. These chemical contaminants pollute water through storm water runoff. When it rains, the water washes these chemicals off the lawns and into water bodies such as streams, rivers, and
16
oceans. The by-product of agricultural activities, urbanization and industrialization result in pollution and degradation of the available water resources (Waziri et al., 2009). Milovanovic (2007) further explains that this increasing surface water pollution causes not only deterioration of water quality, but also threatens human health, balance of aquatic ecosystem, economic development and social prosperity. Some of these chemically derived contaminants that can affect the quality of the water includes the following. a. i.
Chemical elements found in water Aluminium (Al)
Naturally occurring aluminium as well as aluminium salts used as coagulants in drinkingwater treatment are the primary sources of aluminium in drinking-water. It has no known necessary role in human or animal diet. It is nontoxic in the concentrations normally found in natural water. Elevated dissolved aluminium concentrations in some low pH waters can be toxic to some types of fish (Hem, 2013). The presence of aluminium at concentrations in excess of 0.1–0.2 mg/l often leads to consumer complaints as a result of deposition of aluminium hydroxide floc and the exacerbation of discoloration of water by iron. It is therefore important, to optimize treatment processes in order to minimize any residual aluminium entering the distribution system. Under good operating conditions, aluminium concentrations of less than 0.1 mg/l are achievable in many circumstances. Available evidence does not support the derivation of a health-based guideline value for aluminium in drinking-water (WHO, 2011). ii.
Cadmium (Cd)
A cumulative poison, Cadmium is very toxic. The maximum permissible contamination level is 5 µg/l. It is not known to be either biologically essential or beneficial, but can promote 17
renal arterial hypertension. Elevated concentrations may cause liver and kidney damage, or anaemia, retarded growth, and death (USEPA, 2014). iii.
Copper (Cu)
It is essential to metabolism; copper deficiency in infants and young animals results in nutritional anaemia. The action level of copper is 1,300 µg/l. Large concentrations of copper are toxic and may cause liver damage. Moderate levels of copper (near the action level) can cause gastro-intestinal distress. If more than 10% of samples at the tap of a public water system exceed 1,300 µg/l, the USEPA requires treatment to control corrosion of plumbing materials in the system (USEPA, 2014). Staining of sanitary ware and laundry may occur at copper concentrations above 1 mg/l. At levels above 5 mg/l, copper also imparts a colour and an undesirable bitter taste to water. (Copper can give rise to taste, and should not exceed guideline value of 2 mg/l in drinking water (WHO, 2011). iv.
Iron (Fe)
Iron forms rust-colour sediment and stains laundry, utensils, and fixtures reddish brown. Secondary maximum contamination level is 300 µg/l. It is objectionable for food and beverage processing, and can promote growth of certain kinds of bacteria that clog pipes and well openings (USEPA, 2009). v.
Lead (Pb)
This is a cumulative poison, toxic in small concentrations. It can cause lethargy, loss of appetite, constipation, anaemia, abdominal pain, gradual paralysis in the muscles, and death. If 1 in 10 samples of a public water supply exceed 15 µg/l, the USEPA recommends treatment to remove lead and monitoring of the water supply for lead content (USEPA, 2014).
18
vi.
Lithium (Li)
It is reported as probably beneficial in small concentrations (250-1,250 µg/l); help strengthen the cell wall and improve resistance to genetic damage and to disease. Lithium salts are used to treat certain types of psychosis. vii.
Manganese (Mn)
At levels exceeding 0.1 mg/l, manganese in water supplies causes an undesirable taste in beverages and stains sanitary ware and laundry. The presence of manganese in drinkingwater, like that of iron, may lead to the accumulation of deposits in the distribution system. Concentrations below 0.1 mg/l are usually acceptable to consumers. Even at a concentration of 0.2 mg/l, manganese will often form a coating on pipes, which may slough off as a black precipitate. The health-based value of 0.4 mg/l for manganese is higher than this acceptability threshold of 0.1 mg/l (WHO, 2011). viii.
Mercury (inorganic) [Hg]
No known essential or beneficial role in human or animal nutrition. Maximum contamination level is 2 µg/l. Liquid metallic mercury and elemental mercury dissolved in water are comparatively nontoxic, but some mercury compounds, such as mercuric chloride and alkyl mercury, are very toxic. Elemental mercury is readily alkylated, particularly to methyl mercury, and concentrated by biological activity. Potential health effects of exposure to some mercury compounds in water include severe kidney and nervous system disorders (USEPA, 2014).
19
ix.
Nickel (Ni)
It is very toxic to some plants and animals. Toxicity for humans is believed to be very minimal (USEPA, 2014). x.
Selenium (Se)
The permissible maximum contamination level for this metal is 50 µg/l. It is essential to human and animal nutrition in minute concentrations, but even a moderate excess may be harmful or potentially toxic if ingested for a long time. Potential human health effects of exposure to elevated selenium concentrations include liver damage (USEPA, 2014). xi.
Silver (Ag)
It causes permanent bluish darkening of the eyes and skin. Its secondary maximum contamination level is 100 µg/l. When found in water, it is almost always from pollution or by intentional addition. Silver salts are used in some countries to sterilize water supplies. It is toxic in large concentrations (USEPA, 2014). xii.
Sodium (Na)
The taste threshold concentration of sodium in water depends on the associated anion and the temperature of the solution. At room temperature, the average taste threshold for sodium is about 200 mg/l. No health-based guideline value has been derived, as the contribution from drinking-water to daily intake is small (WHO, 2011). xiii.
Zinc (Zn)
It is essential and beneficial in metabolism; its deficiency in young children or animals will retard growth and may decrease general body resistance to disease. Zinc imparts an undesirable astringent taste to water at a taste threshold concentration of about 4 mg/l (as 20
zinc sulfate). Water containing zinc at concentrations in excess of 3–5 mg/l may appear opalescent and develop a greasy film on boiling. Although drinking-water seldom contains zinc at concentrations above 0.1 mg/l, levels in tap water can be considerably higher because of the zinc used in older galvanized plumbing materials; this may also be an indicator of elevated cadmium from such older (WHO, 2011). b.
Physical and Chemical indicator tests
i.
pH
pH is a measure of the hydrogen ion concentration; pH of 7.0 indicates a neutral solution while a pH values lower than 7.0 indicate acidity and pH values higher than 7.0 indicate alkalinity. pH standard of drinking water is 6.5-8.5 units. Water generally becomes more corrosive with decreasing pH; however, excessively alkaline water may also be corrosive (USEPA, 2009). Although pH usually has no direct impact on consumers, it is one of the most important operational water quality parameters. Careful attention to pH control is necessary at all stages of water treatment to ensure satisfactory water clarification and disinfection. For effective disinfection with chlorine, the pH should preferably be less than 8; however, lower pH water (approximately pH 7 or less) is more likely to be corrosive (WHO, 2011). ii.
Dissolved Oxygen
Dissolved oxygen is required by higher forms of aquatic life for survival. Measurements of dissolved oxygen are used widely in evaluations of the biochemistry of streams and lakes. Oxygen is supplied to ground water through recharge and by movement of air through unsaturated material above the water table (Moniruzzaman et al., 2013). The dissolved
21
oxygen content of water is influenced by the source, raw water temperature, treatment and chemical or biological processes taking place in the distribution system. Depletion of dissolved oxygen in water supplies can encourage the microbial reduction of nitrate to nitrite and sulfate to sulfide. It can also cause an increase in the concentration of ferrous iron in solution, with subsequent discoloration at the tap when the water is aerated. No health-based guideline value is recommended. However, very high levels of dissolved oxygen may exacerbate corrosion of metal pipes (WHO, 2011). iii.
Nitrite plus nitrate
Concentrations nitrite and nitrate greater than local background levels may indicate pollution by feedlot runoff, sewage, or fertilizers. Concentrations greater than 10 mg/L, as nitrogen, may be injurious when used in feeding infants (USEPA, 2014). iv.
Phosphorus
Dense agal blooms or rapid plant growth can occur in waters rich in phosphorus. It is a limiting nutrient for eutrophication since it is typically in shortest supply. Sources of Phosphorus are human and animal wastes and fertilizers (USEPA, 2014). v. Specific conductance It is a measure of the ability of water to conduct an electrical current it varies with temperature, with the magnitude depending on concentration, kind, and degree of ionization of dissolved constituents. It can be used to determine the approximate concentration of dissolved solids. Conductance values are reported in microsiemens per centimeter at 25°C (USEPA, 2014).
22
vi.
Temperature
This affects the usefulness of water for many purposes. Generally, users prefer water of uniformly low temperature. Temperature of ground water tends to increase with increasing depth to the aquifer. The temperature of the water body is inversely proportional to the dissolved oxygen. vii.
Hardness and noncarbonated hardness
This is related to the soap-consuming characteristics of water. Presence results in formation of scum when soap is added. May cause deposition of scale in boilers, water heaters, and pipes. Hardness contributed by calcium and magnesium, bicarbonate and carbonate mineral species in water is called carbonate hardness; hardness in excess of this concentration is called noncarbonated hardness. Water that has a hardness less than 61 mg/l is considered soft; those with 61-120 mg/l, moderately hard; while those with 121-180 mg/l are hard; and more than 180 mg/l, very hard (Heath, 1983). viii. Alkalinity It is a measure of the capacity of unfiltered water to neutralize acid. In almost all natural waters, alkalinity is produced by the dissolved carbon dioxide species, bicarbonate and carbonate. It is typically expressed as mg/l CaCO3 (USEPA, 2009). ix.
Dissolved solids
This is the total of all dissolved mineral constituents, usually expressed in milligrams per liter. The concentration of dissolved solids may affect the taste of water. The secondary maximum level is 500 mg/l. Water that contains more than 1,000 mg/l is unsuitable for many industrial uses. Some dissolved mineral matter is desirable, otherwise the water would have
23
no taste. The dissolved solids concentration is commonly called the water’s salinity and is classified as follows: Fresh, 0-1,000 mg/l; Slightly saline, 1,000-3,000 mg/l; Moderately saline, 3,000-10,000 mg/l; Very saline, 10,000-35,000 mg/l; and Briny, more than 35,000 mg/l (Heath, 1983). x. Basic Cations (Ca, Na, K and Mg) Calcium and Magnesium are the major cause of hardness and scale-forming in water (USEPA, 2014). Large concentration of Sodium and Potassium may limit use of water for irrigation and industrial use and, in combination with chloride, give water a salty taste. Abnormally large concentrations of these cations may indicate natural brines, industrial brines, or sewage (USEPA, 2014). The Sodium-adsorption ratio (SAR) is used to express the relative activity of sodium ions in exchange reactions with soil. It is important in irrigation water; the greater the SAR, the less suitable the water for irrigation (USEPA, 2014). xi.
Anions (HCO3, SO42-, halides, Nitrite/nitrate)
Bicarbonate in combination with calcium and magnesium is responsible for hardness in water (USEPA, 2014). Sulfates of calcium and magnesium form hard scale. Its maximum permissible concentration level is 250 mg/L. Large concentrations of sulfate have a laxative
24
effect on some people and, in combination with other ions, give water a bitter taste (USEPA, 2014). Chloride, in large concentrations increase the corrosiveness of water and, in combination with sodium, give water a salty taste. The maximum allowable concentration level is 250 mg/l (USEPA, 2009). Among the halides anions, fluoride and bromide are of much concern. Fouride ion reduces incidence of tooth decay when optimum fluoride concentrations is present in water consumed by children during the period of tooth calcification. Maximum allowable concentration level is 4.0 mg/l. Potential health effects of long-term exposure to elevated fluoride concentrations include dental and skeletal fluorosis (USEPA, 2014). Bromide is not known to be essential in human or animal diet, nor does it have any ecologic significance when it occurs in small concentrations typically found in fresh waters of the United States (USEPA, 2014). On the other hand, iodide is essential and beneficial element in metabolism; deficiency can cause goitre (USEPA, 2014). Nitrite (mg/l as N) is commonly formed as an intermediate product in bacterially mediated nitrification and de-nitrification of ammonia and other organic nitrogen compounds. It is an acute health concern at certain levels of exposure. Nitrite typically occurs in water from fertilizers and is found in sewage and wastes from humans and farm animals. Concentrations greater than 1.0 mg/l, as nitrogen, may be injurious when used in feeding infants (USEPA, 2009).
25
xii.
Other inorganics
Ammonia is a plant nutrient that can cause unwanted algal blooms and excessive plant growth when present at elevated levels in water bodies. Sources include decomposition of animal and plant proteins, agricultural and urban runoff, and effluent from waste-water treatment plants (USEPA, 2014). Organic species are unstable in aerated water and generally are considered to be indicators of pollution through disposal of sewage or organic waste (Hem, 2013). Nitrogen in reduced (ammonia) or organic forms is converted by soil bacteria into nitrite and nitrate, a process referred to as nitrification (USEPA, 2014).
2.4
Water Pollution and Quality of Fish
Good water conditions are necessity for the survival and growth of fish since the entire life process of the fish wholly dependent of the quality of its environment. The quality of water in terms of its suitability for fish growth, and its fertility, to a large extent determines the rate of growth of the fish. A river water that is poor in quality will endanger the health of the fish (Bolorunduro and Abdullah, 1996). Water bodies get dirty due to pollution by human activities. This water pollution affects all kinds of aquatic flora and fauna severely proves lethal to them. Pollution from sewage and human waste introduce pathogens into the water sources resulting in infections and death of aquatic inhabitants (Ganguly, 2013). Water pollution imposes this adverse effect on fishes are mainly affected from the human nuisances (Cruickilton and Duchrow, 1990). Fluctuations in water temperature from power plants and factories kill off aquatic life especially fish and this make them to migrate for relocation in an attempt to find waters with a more sustainable thermal condition (Ganguly et al., 2011). Excessive noise production from boats and drilling has also been reported by Ganguly (2013) 26
to cause stress on fish and other marine life which make them sick and lethargic and thus affects their mating behaviour adversely. Kivi (2010) reported that radioactive waste generated from industrial wastes enter the water bodies and are absorbed by fish and can cause genetic, mutagenic and teratogenic defects in them.
2.5
Prevention of Water Pollution
Prevention, treatment, and remediation are employed in solving water quality problems. Pollution is prevented before it enters waterways, or wastewater treated before discharge, or biological integrity restored through remediation. Among the main source of pollution is wastewater discharge from human waste transport and industrial and agricultural use into water body, which can be controlled in one of the under-listed ways: actions at the point of generation; pre-treatment of wastewater prior to discharge to municipal systems or local waterways; and Complete treatment and reuse. Dealing with “non-point source” pollution is the most difficult water-quality challenges. Non-point source pollution is the result of precipitation runoff from many diffuse sources including fertilizers, nutrients, and pesticides from agriculture; and oil, grease, and toxics from urban settlements, these are not easily regulated. Pollution prevention at its source, in industry, agriculture, and human settlements, is often the cheapest, easiest, and most effective way to protect water quality. In an industrial setting, this is called cleaner production. Prevention pollution is the reduction or elimination of wastes from the source which reduces
27
or eliminates the use of hazardous substances, pollutants, and contaminants and this can be accomplished by: i.
formulating products that produce less pollution and need less resources during their manufacture and use;
ii.
reducing the use of toxic materials for pest control, nutrient application, and water usage;
iii.
reducing the amount of hazardous materials used and disposed and reducing wastewater production;
iv.
modifying equipment or technologies so that they generate less waste;
v.
implementing better training, maintenance, and housekeeping so that leaks and fugitive releases are reduced; and
vi.
Reducing water consumption.
Pollution prevention is targeted at reducing the overall generation of waste, and an important advantage of pollution prevention over pollution control is that prevention protects all environmental media (air, water, and land) simultaneously, while the control may shift waste from one medium to another (e.g., an air pollution scrubber can send air contaminants into water). Preventing pollution can turn waste streams into valuable resource streams. Pollution prevention gets at the root causes of pollution, namely waste and inefficiency. Preventing pollution means less money spent on waste handling, storage, treatment, remediation, and regulatory monitoring (UNEP, 2010).
28
2.5.1 Source Water Protection If source water is protected, it serves as a key to improving water quality and decreasing treatment costs focuses on protecting the sources of vital drinking water supplies from contamination in order to reduce or eliminate the need for treatment. Formally, approach to water management involves treating water at many stages to remove contaminants and the environmental and economic costs of this strategy are high, particularly as energy costs rise. Healthy, resilient ecosystems help purify and regulate water, thereby avoiding pollution entering into waterways (UNEP, 2010).
2.5.2 Industrial Point-Source Pollution Industrial releases are point sources which are often regulated. Thus, it is easier to characterize the quality of existing effluent, and there are greater regulatory and financial incentives for industries to prevent pollution and reduce costs. Cleaner production efforts have been supported through United Nation programmes, including UNEP and other organizations. In 1994, the United Nations Industrial Development Organization (UNIDO) along with United Nations Environmental Programme (UNEP) established the National Cleaner Production Centres Programme (NCPCs), which helps developing countries and countries with economies in transition to incorporate cleaner production into their industrial development and environmental legislation, and undertake activities to support cleaner production (UNEP, 2010). Cleaner production as a strategy, is to increase the efficiency of use of raw materials, energy, and water and reduce sources of waste and emissions and it can take place in a number of ways:
29
• Reduction or elimination of the use of solvents in industrial processes; • Reduction or elimination of the use of toxic chemicals in processes; • Reduction in overall water use in the system; and • Closure of water cycle within industries and eliminating wastewater discharge (UNEP, 2010).
2.5.3 Agricultural Non-Point Source Pollution Pollution due to agricultural activities around the world contribute significantly to waterpollutant loads. Nitrogen, phosphorus, pesticides, and sediment, which impair both surface and groundwater usually form the contaminants of agricultural runoff. There are several ways to reduce the impacts of agriculture on water quality, and scales of intervention from the farm level to the state. i.
Farm level At the farm level, the use of organic, or biological, agriculture are innovations which decreased the need for chemical inputs and a movement away from synthetic chemicals in favour of crop rotation, mulching, composting, cover cropping, and integrated pest management. Crop rotation helps to avoid depletion of soil nutrients. In addition, less intensive tillage practices that leave varying amounts of crop residue or mulch on the soil have been shown to increase soil organic matter without the use of synthetic fertilizers. Cover cropping with clover, ground nut, vetch, and other nitrogen-fixing plants is another practice that enhances soil health without the use of chemicals. Also, composting plant matter and animal manure produces a rich soil that can be applied to fields. To reduce pesticides input, the use of beneficial insects, fungi, or bacteria that will eat or negatively affect the pests are 30
employed. In rare cases chemicals may be used, but they are targeted to particular types of insects – rather than broad- spectrum pesticides that can affect many insect species beyond those of concern (UNEP, 2010). ii.
Basin level At the basin level, the types and locations of different land uses is usually considered to control agricultural water runoff particularly steep slopes which facilitate the runoff of water, sediment, and chemicals from agricultural lands. Contour farming and terracing can decrease erosion and runoff from agricultural fields and are critical for pollution prevention on steep slopes (UNEP, 2010). iii.
National and state/provincial level
Conclusively, it is essential that states set standards for agricultural practices and runoff pollutant levels and that they should be able to enforce them through monitoring, measurement, and fines or other consequences for violations. In addition to enforcement, state outreach and assistance programmes can help farmers to implement innovative practices and can also provide financial incentives for adoption of farming techniques that use fewer inputs, and provide grants or loan programmes for upgrading infrastructure and installing more efficient irrigation systems (UNEP, 2010).
2.5.4 Settlements Natural hydrologic processes can reduce aquifer recharge by reducing recharge areas. The many impervious surfaces in cities – streets, roofs, parking lots, sidewalks – prohibit water from filtrating into the ground and result in large quantities of urban runoff. This runoff collects pollutants as it flows across city surfaces.
31
The significance of polluted runoff to water quality problems highlights the link between land use and water quality and the need to better integrate water quality concerns into development and land-use planning and policies (UNEP, 2010).
2.6
Treatment of Water Pollution
Treatment of water pollution to improve quality for drinking and other purposes became inevitable when efforts to prevent pollution from entering water sources are ineffective or insufficient. It is also necessary to treat the wastewater after it has been used for these purposes. There are technological solutions to treat water quality to particular standards as well as ecological systems that purify and improve water quality (UNEP, 2010).
2.6.1 Drinking Water Treatment Treating drinking water for consumption can be carried out at the municipal level, the community level, or at the household level. Different technologies exist for drinking water treatment at each of these scales. Developed countries often provide treated drinking water that is easily accessible at a household tap whereas in many developing countries, limited water is available from the municipality via individual household taps. Even those that access to individual household connections need to treat this water before consumption (UNEP, 2010). i. Municipality Source of drinking water can be from groundwater, rivers, lakes, canals, reservoirs, and even from seawater. The water is transported from the source, and is treated to ensure that it is suitable to drink by improving the physical, chemical, and biological characteristics of the water. 32
Water purification can involve a series of processes depending on the source water quality. Screening is performed for large debris, pre-conditioning to treat hardness and normalize pH, then flocculation to clarify the water by binding particles, settling the particles, and filtration to remove additional suspended particles and microbiological contaminants. Disinfection is the final phase which at a municipal scale uses chlorine or chlorine-based disinfectants which leave a residual at the tap. ii. Community This include community- scale filtration or disinfection plants that provide safe drinking water from existing sources. An example of this is the Water Health International model, where water is filtered and disinfected using ultraviolet technology and the water is then sold to residents or businesses for use in drinking and use in commercial operations. In some cases, Water Health Centre is installed, and then payment for ongoing service charges for the water used. Their average Centre is designed to provide a community of 3,000 residents with up to 20 litres of drinking water per person per day (UNEP, 2010). Community-scale interventions for pure drinking water are also used in emergency situations and in transition scenarios. Drinking water treatment can also be done through packed lowpressure reverse osmosis units (UNEP, 2010). iii. Household Recently there is an increasing focus on the importance of drinking water treatment at the household level. While, ideally, every person should have access to safe drinking water from a household tap, it has become clear that strategies to improve the quality of drinking water through household water treatment and appropriate storage could have a significant impact.
33
UV disinfection, chlorine, or boiling is considered an effective way of treating water at the Point-of-use or treating water in the home before it is consumed. This pinpoint the importance of improving water quality in improving health. Amongst the numerous strategies in use to provide on-site or household water treatment includes: from low-cost and small-scale chlorine disinfection systems, ceramic filters, flocculation/ disinfection products, solar disinfection, and household boiling to very expensive on-site systems using reverse osmosis systems, which can be either energy- or water- intensive depending on the type of system. These household treatment systems for drinking water are solving access to safe water for individual users, rather than municipalities, regions, or villages (UNEP, 2010).
2.6.2 Treatment for Other Uses i. Agricultural use Water for agricultural use can be of much lower quality than drinking water or even industrial water, in many cases. Often, this water may be treated using traditional techniques such as those described for municipal drinking water, or through bioremediation. Increasingly, agricultural areas recycled human wastewater as a new, drought-proof water source. In some countries, regulations have been set to ensure that recycled water is safe for agricultural use. In addition, sustainable agriculture and forestry are increasingly seen as ways to protect source waters since they both are extensive, pervious land uses that allow groundwater recharge and are less disruptive to natural hydrology than urban areas. However, it is critical that chemical inputs are minimized in order to avoid ground- and surface-water contamination (UNEP, 2010).
34
ii. Industrial use Industries require a particular standard of water quality for input into production processes. Water purification may be needed to meet the standard requirements for pharmaceuticals, computer parts, high-grade chemical products, processed foods, or other industrial products. Biological, physical, and chemical processes are water purification methods that are used by industries. These particular treatment processes are applied to surface, ground, or municipally supplied water to meet the standard required for particular processes. In some areas, industries may use treated wastewater from municipal wastewater utilities, which is often of much higher quality than any existing or available surface or groundwater sources (UNEP, 2010).
2.7
Wastewater Treatment
Centralized municipal-level systems (i.e., large systems that treat wastewater from many users at one site) or decentralized systems (i.e., those that treat individual homes or businesses or small groups of individual users) can be used in wastewater treatment. In centralized systems the water is usually discharge to surface waters, whereas decentralized systems can produce water for local reuse, release to the soil or local surface water. In many cases, urban wastewater treatment in industrialized nations has been conducted at centralized facilities where the industrial wastewater is typically treated on-site. In “developing” nations, centralized systems are insufficient, unreliable, or simply absent and the wastewater of many local communities is simply discharged directly into waterways. Over 80 percent of the sewage in developing countries is discharged untreated in receiving water bodies (UN WWAP, 2009; UNEP, 2000).
35
Decentralized systems provide a cheaper alternative to centralized systems, however, they are more prone to being poorly designed, have less oversight, and often serve as major source of groundwater contamination if they do not adequately treat wastewater (UNEP, 2010).
2.7.1 Domestic Wastewater Treatment i. Municipal Municipal wastewater consists of human wastes from toilets, washing facilities, kitchens, and other typical household water uses. It also includes commercial wastewater and some from industries. Wastewater quality is compromised physically (e.g., colour, odour, temperature, etc.); chemically (e.g., biochemical oxygen demand, total organic carbon, etc.); and biologically (e.g., microbiological contaminants like coliforms, pathogens, viruses). Physical, chemical, and biological processes are used to treat these water quality parameters, which result in treated effluent and solid waste or sludge (UNEP, 2010). Physical water treatment technologies, rely on separating or filtering contaminants from wastewater, or destroying those contaminants, using mechanical systems. Filtration is achieved by running contaminated waters through fine grates or using reverse osmosis systems that separate often very small contaminants from water. Sedimentation, the process of letting suspended solids settle at the bottom of a holding area, has long been used to allow for easier removal of contaminants. Water is stirred mechanically to promote coagulation, which also makes contaminants easier to remove through subsequent filtering or settling processes. Other physical methods include boiling/incineration and irradiation which can also disinfect (i.e., remove or neutralize certain pathogens) wastewater (UNEP, 2010).
36
Chemical water-treatment technologies rely on introducing chemicals that break apart, neutralize, or aggregate contaminants. Chemical solutions are able to “clean” small pollutants – such as nutrients like nitrates and phosphates, as well as microorganisms – from wastewaters that are not captured using physical treatment methods. Chemical treatments often use either disinfection or coagulation/ flocculation to clean wastewater. Disinfection is the treatment of effluent using chemicals to destroy pathogens. Historically, the most common disinfecting agent used in water treatment has been chlorine, however a variety of chemicals, such as aluminium and iron salts, ozone, and UV-light, can be used. Coagulation and flocculation is the process of destabilizing contaminants to allow them to bind smaller contaminants into larger aggregates to make them easier to separate physically (UNEP, 2010). Biological solutions rely on the natural processes of living organisms – such as microbes or plants – to treat wastewater. For example, trickling filters consist of a fixed bed of materials, such as rock, peat moss, or polyurethane foam covered with a film of microbial growth that cleans contaminants through absorption and adsorption. Activated sludge methods use microorganisms to convert carbon found in wastewater into carbon dioxide and water or to adjust nitrogen levels. Wastewater treatment systems are also increasingly incorporating outdoor “constructed wetlands” that use plant systems to break down contaminants before they are released to natural water bodies (UNEP, 2010). ii. Community Community wastewater treatment come to effect when there are very disperse ex-urban, periurban, or rural populations. Due to the fact that the populace live in disperse settlement it
37
makes sewage collection, transport, and centralized wastewater treatment difficult. This often exists in many parts of the developing world because of a lack of resources and government investment. In the case of less dense settlements, or those that are far from centralized sanitation systems, community-level systems can be effective at treating wastewater before it is disposed of in surface water. In community-level wastewater systems physical, chemical, and biological treatment is also use but on a smaller scale when compared with municipal wastewater treatment. On-site systems can be low-energy and low-cost systems for water collection, storage, disinfection, and waste treatment. Examples of these include ecosanitation approaches and traditional septic tanks. Conventional mini-water plants (using, for example, reverse osmosis or ultraviolet technologies) and wastewater treatment plants (e.g. membrane bio-reactors) are expensive and energy intensive on-site systems that can be used. For high-quality treatment at low cost and at much smaller scale membrane bioreactors are available. And microfiltration, reverse osmosis, electro-dialysis, and advanced technologies make it possible to treat small, intermittent water flows that are not easily treated with biological processes like activated sludge or membrane bioreactors. (UNEP, 2010). iii. Household Human wastewater can be treated on- site at a household level and this form of treatment is most appropriate for rural areas or dispersed settlements for public health reasons. Household sanitation usually take the form of a dry or a wet toilet.
38
Septic tanks are a usual method of household wastewater treatment from a pour flush toilet. A septic tank is a watertight chamber where wastewater, both black water from the toilet and grey water from washing from the household is conveyed. The two chambers are used to settle out solids and provide space for anaerobic processes to reduce solids and organic materials. Treatment is completed only if the accumulated sludge in a septic tank is independently removed and dried. There are numerous dry toilet systems (Morgan, 2007), some of which may be shallow toilets where a tree is planted and grown after short-term use, or deep composting toilets where humic material is reused for horticulture. The overall concept of many of these toilets is part of the ecosanitation model which is based on closing the nutrient loop in sanitation and moving away from conventional waterborne sanitation. Countries facing water scarcity usually use ecosanitation. In addition, a core tenet of ecosanitation is that human excreta contains valuable nutrients that can be used to help enhance food security when treated and handled properly (UNEP, 2010).
2.7.2 Industrial Wastewater Treatment Industrial processes usually generate significant amounts of wastewater which if not prevented or recycled on-site, needs to be treated before disposal. Standards for industrial effluent quality are in place in many parts of the world, but in many places it is either not adequate or appropriately enforced. If the industrial effluent water quality is severely degraded or toxic, the industrial facility owner should be responsible for safely removing pollutants from the water before discharge and appropriately disposing of the hazardous sludge. However if the industrial wastewater is not hazardous, it can be treated at the municipal wastewater treatment facility. 39
Since solvents, paints, pharmaceuticals, pesticides, and synthetic organic products are difficult to treat in wastewater, the goal should therefore be to reduce or eliminate the use of these products, or other appropriate non-waterborne methods of disposal be used. Advanced oxidation processing, distillation, adsorption, vitrification, and incineration are among the methods to treat synthetic organics. Many toxic organic materials and heavy metals like cadmium, chromium, zinc, silver, thallium, arsenic, and selenium are also difficult to treat in industrial wastewater (UNEP, 2010).
2.7.3 Agricultural Wastewater Treatment Agricultural wastewater, or runoff, is collected to be reused or disposed of. Often, water of extremely poor quality need to be treated before it can be reused or discharged. Normally, the water does not need to meet drinking water standards to be re-applied to a field or discharged into a waterway as such less intensive treatment options are chosen since. Bioremediation is one form of treatment whereby plants, microorganisms, fungi, or their enzymes are used to filter and remove contaminants from polluted waters (UNEP, 2010). Wetlands create anoxic environments that encourage de-nitrification and this is useful in terms of the removal of nitrates, a common component of agricultural runoff.
2.8
Waterborne Diseases
Water-borne infections is one of the most serious environmental health problems faced today. Most people living in developing countries do not have access to clean water (CDC, 2014) rather use contaminated water for domestic and other uses. This predisposes them to various diseases and death.
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In Nigeria and Africa in general, several waterborne diseases are very common (MWRMD, 2013), so it is very important to understand what causes them, how do they transmit, what are the dominant symptoms, and especially how to diagnose, treat and prevent them (HAID, 2015). Although the symptoms can be mild, they can be life-threating as well, and today water-borne diseases are the second leading cause of death globally in children under the age of five (HAID, 2015).
2.8.1 Typhoid Fever Typhoid fever is caused by a bacterium known as Salmonella typhi (CDC, 2014). It is excreted in the stools of infected individuals and predominantly transmitted through contaminated water, flies can also play a role as vectors contaminating food (W.D. I, 2014). There is usually 1 to 3 weeks incubation period after the infection and before the symptoms of, disease starts gradually with headache, fever and abdominal pain (CDC, 2014). There is usually constipation which is then followed by diarrhoea (W.D.I, 2014). In severe forms of the disease, body temperature can rise up to 40° C and haemorrhage can occur. In such cases death rate can reach up to 30% (CDC, 2014). As such, the use of antibiotics early in the infection is crucial to prevent life-threatening complications (CDC, 2014). Although there is an available vaccine for this disease, it unfortunately provides only limited protection (MWRMD, 2013).
2.8.2 Dysentery Two common cause of dysentery are Shigella spp., and Entamoeba histolytica. Shigella belongs to Enterobacteriaceae while Entamoeba histolytica is an amoeba (CDC, 2014). Shigella is present in the stools of infected persons and is readily transmitted to human 41
through contaminated water to cause a disease (W.D.I, 2014). Main signs and symptoms of the Shigella infection fever, cramps in their abdomen, pain in the rectum and bloody diarrhoea (CDC, 2014). In a severe infection, high fever may induce seizures in children younger than 2 years old, and people with weakened immune systems are at special risk, therefore there is sneed to treat them with antibiotics (CDC, 2014). Entamoeba histolytica is prevalent in regions where human stool is used as fertilizer (MWRMD, 2013) and it is spread via raw fruit and vegetables irrigated with contaminated water. Most of infections with this organism are asymptomatic, but when present they are predominantly diarrhoea, nausea, weight loss and tenderness in the abdomen (CDC, 2014). Rarely, the disease progresses and perforates the intestine and spread to liver, which can be life-threatening (HAID, 2015).
2.8.3 Cholera Vibrio cholerae is the causative organism of cholera (CDC, 2014) and infected individuals can excrete it and contaminate water for at least 3 weeks after the illness begins (HAID, 2015). It can also survive in seafood (MWRMD, 2013). Symptoms of the bacterium infection are heavy diarrhoea and vomiting with considerable loss of fluids, leading to for severe dehydration which can lead to death (CDC, 2014). Treatment is usually by urgent replacement of lost fluids (W. D. I, 2014). Although managing patients with acute watery diarrhoea is similar regardless of the cause, it is important to recognize cholera because of its potential for a wide-spread outbreak (MWRMD, 2013).
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2.8.4 Viral Gastroenteritis Novoviruses and rotaviruses are leading causes of waterborne viral gastroenteritis, however, astroviruses, sapoviruses and enteric adenoviruses are also common (CDC, 2014; W.D.I, 2014). This type of gastroenteritis is also commonly known as “stomach flu”. Infection with these contagious viruses causes fever, stomach cramps, sudden vomiting and watery diarrhoea (HAID, 2015). The incubation period is less than 2 days and it is most often spread within families and closed places like schools and day-care centers. The major source of infection is contaminated water (MWRMD, 2013). In this type of infections, antibiotics are not useful, affected individuals are treated by urgent replacement of water and minerals lost with diarrhoea – especially in the case of new-borns and the elderly (CDC, 2014). In most cases, microbiological identification to identify the specific virus is important for public health and infection control purposes (W. D.I, 2014).
2.8.5 Prevention of Waterborne Diseases There are several general advice which should be followed in order to avoid aforementioned waterborne diseases, regardless of what microorganism is responsible (Oguntoke et al., 2009; Awojobi, 2011; W. D. I, 2014). i.
Do not drink untreated water.
ii.
Addition of 1 teaspoon of household bleach to 20 litres of water and leaving it for at least 1 hour before drinking.
iii.
Boil water for at least 3 minutes, and allow to cool before drinking.
iv.
Using clean drinking water containers.
v.
Washing fruit and vegetables with clean water before eating.
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vi.
Strict adherence to hygiene after going to the toilet and before preparing food.
vii.
Washing of clothes and utensils about 30 meters away from drinking sources.
viii.
Adhering to good waste management practices, such as collection and disposal of faecal matter.
ix.
Rinse raw produce (fish) thoroughly under running tap water before cooking.
x.
Scrub containers and utensils used in handling uncooked fish before using with ready-toserve foods.
xi.
Don’t taste raw fish or shellfish.
xii.
Refrigerate or freeze raw/cooked fish leftovers in small, covered shallow containers within two hours after cooking. In developing countries with inadequate infrastructure it is hard to get rid completely of water-borne diseases (Oguntoke et al., 2009; Awojobi, 2011). Application of these general principles constantly and with incessant effort water-borne diseases can be put under a control, so prevention is still the best way to minimize their detrimental effect on the human health (Oguntoke et al., 2009).
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CHAPTER THREE
3.0
MATERIALS AND METHODS
3.1
Study Area
River Kaduna got its name from the Hausa word for crocodiles that lived in the river and its surrounding area. The lower part of River Kaduna called River Lavun (also known as River Wuya) passes through Wuya Kpata, near Bida, Lavun Local Government Area of Niger State, North Central Nigeria. It flow into River Niger which flows for 550Km through Nigeria. It has a confluence with River Niger at Muregi near Patigi in Kwara State, Nigeria. River Lavun forms the eastern border of Local Government Area. The coordinates of the River Lavun at Wuya Kpata is 9°08'21"N 5°49'57"E. River Lavun is the main source of water in Wuya Kpata and environ and it is used for many purposes such as drinking, irrigation, fishery, industrial processes, transportation and waste disposal. Its climate is tropical with strong seasonal rain fall between April and October and a dry season between the months of November and March. Fig. 3.1 shows the schematic diagram of the study area of River Lavun. Samples were collected at three points designated A, B and C. Point A is about 400m before the settlement, Wuya Kpata, and Point C is about 400m after the settlement while Point B is at settlement.
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Fig. 3.1: Map of study area showing sampling points on River Lavun. 46
3.2. Materials 3.2.1 Test Samples Water samples from designated points of River Lavun in the study area. Fish samples collected from the River Lavun: Clarias gariepinus (cat fish) was the only species of fish that was collected. Cat fish belongs to the Family Claridae. It lacks scales and are usually very long, 0.03m to 4.5m (Chambers, 2008). The fish can stay alive for some hours outside water.
3.2.2 Reagents and Diagnostics They were obtained from four main sources. Reagents obtained from Oxoid Ltd, Basingstoke, England are Oxidase test strip, Kovacs reagent, VP1 and VP11 reagent, TDA reagent and Fast Blue reagent. Acridine orange was a product of BDH Chemicals Poole, England while Crystal violet was obtained from May & Baker Ltd, Dagenham, UK and Hydrogen peroxide was a product of SKG Pharma. Ltd, Lagos, Nigeria. MacFarland turbidity standard was prepared in the Laboratory of Department of Pharmaceutics and Pharmaceutical Microbiology, Ahmadu Bello University Zaria, Nigeria.
3.2.3 Culture Media The bacteriological media used were mostly Oxoid products. They included MacConkey Agar, Nutrient Agar, Eosine Methylene Blue (EMB), Mueller-Hinton Agar, Mannitol Salt Agar, Aesculin-Azide Agar, Azide dextrose Broth, Plate count Agar, Salmonella-Shigella Agar, Microbact GNB 12E and Microbact Staphylococcal 12S. Nutrient Broth and Peptone were however products of Fluka, Biochem, U.S.A. 47
3.2.4 Equipment
Atomic Absorption Spectrophotometer, Model AA240FS, Varian, Australia.
Autoclave, Adelphi MFG company Ltd, Portland
Binocular Microscope, Wild M11, Swizerland
Centrifuge, Model 5417R, Eppendorf, USA
Colony counters, Model 630, NAPCO Portland, Oregon, USA
Conductivity meter, Model 4010, Jenway, UK
Electronic weighing balance, Model PA313,Ohaus, USA
Gel electrophoresis machine, Model HU10, serial no 5237, Max Fill Scie-plas
Heating mantle, Gallenkamp, England
Hot-Air-Oven, Baird and Tatlock Ltd, London
Incubator, Model 630, Natural appliance: Aheinicke Company Portland, Oregun USA
pH Meter, Model 3505, Jenway, UK
UV- visible Spectrophotometer, Model 6405, Jenway, UK.
3.2.5 Antibiotic Discs The following antibiotic sensitivity discs were obtained from Oxoid Ltd., Basingstoke, UK: Chloramphenicol (30 µg), Gentamicin (10 µg), Ciprofloxacin (5 µg), Erythromycin (15 µg), Tetracycline (30 µg), Ampicillin (10 µg), Trimethoprim/Sulphamethoxazole (25 µg), Amoxicillin-clavulanate (30 µg), Cefuroxime sodium (30 µg) and Nitrofurantoin (300 µg).
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3.3
Methods
3.3.1 Collection of water samples
Water sample for Microbiological Analysis
Water samples for microbiological analysis were collected in a sterile amber coloured glass bottle. At each sampling point, the bottle was opened and immersed in the river to a depth of about 30cm with its mouth facing the water current, filled to three quarters its capacity and quickly stoppered to avoid contamination. The water was then transported on ice to the Laboratory of the Department of Microbiology, Federal University of Technology (FUT), Minna. Bacteriological analyses of the samples commenced within three hours of collection. This procedure was repeated for each sample. Samples were collected monthly from April to September, 2014.
Water Samples for the Assessment of the Physicochemical Quality and Elemental Analysis
Water samples for determination of physicochemical properties were collected in one litre (1L) capacity plastic containers which had previously been thoroughly washed, rinsed with 50% nitric acid solution and finally rinsed in distilled water. As with microbiological samples, the water was collected but filled to capacity to prevent entrapment of air and promptly closed and transported to the Department of Fishery, Federal University of Technology (FUT), Minna, where the samples were digested for analyses.
3.3.2 Preparation of Water Samples for Elemental Analysis Ten millilitre (10 ml) each of water samples were measured into a beaker and 10 ml mixture of HNO3 – H2O2 (1:1) was added and digested for 2 hours at 160°C. The digest was cooled
49
and filtered and transferred to 100 ml volumetric flask and fill up to the level with de-ionized water (Olaifa et al., 2004). This was used for the elemental analysis.
3.3.3 Collection of Fish Samples Three samples of live C. gariepinus (Cat fish), caught from the river were obtained from vendors monthly, for six months. The fish samples were transported in a jerry can containing the river water, to the Laboratory of the Department of Microbiology, Federal University of Technology (FUT), Minna, where they were cleaned with sterile distilled water. One gram of muscle was cut aseptically, labelled appropriately for microbiological analysis. The remaining portion of the fish were taken to the Department of Fishery, Federal University of Technology (FUT), Minna, for digestion.
3.3.4 Preparation of Fish Samples for Microbiological Analysis One gram (1.0g) each of the fish samples were weighed aseptically, and macerated in 9.0mls of 0.1% peptone water and serially diluted three fold and labelled appropriately (Salihu et al., 2012). This was used for the microbiological analysis. A 1.0 ml from the 10-3 dilution was used for heterotrophic bacterial count while 1.0 ml from the 10-2 dilution was used for faecal coliform count.
3.3.5 Preparation of Fish Samples for Elemental Analysis A 1.0g each of fish muscle was weighed and dissolved in a 10 ml mixture of HNO3 – H2O2 ( 1:1) and digested for 2 hours at 160°C. The digest was cooled and filtered and transferred to 100ml volumetric flask and filled up to the level with de-ionized water (Olaifa et al., 2004). This was used for the elemental analysis.
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3.3.6
Preparation of Bacteriological Media
The dehydrated media were reconstituted with freshly prepared distilled water and distributed into containers as specified by the manufacturers. Except those not required to be sterilized, sterilization of these media was achieved by autoclaving at 121°C for 15 minutes. Prepared media were stored in refrigerators at 4°C until needed.
3.3.7
Heterotrophic (Standard) Plate Count
Heterotrophic plate count was carried out using the pour-plate method as described by America Public Health Association, APHA (1998). Ten fold serial dilution of the water samples was prepared in sterile water. The 10-3 dilution was used. From this, 1ml sample was aseptically transferred into labelled sterile Petri-dishes. Aliquots of 15ml sterile molten Plate Count Agar was then poured into the plates and properly mixed to ensure effective even distribution of the water samples in the agar media. The plates were allowed to set (solidify) and thereafter, placed in incubators at 37°C. The number of colony forming units were counted after an incubation period of 48 hours. The values were multiplied by the dilution factor to calculate the actual microbial levels.
3.3.8
Determination of Faecal Coliform Count
Ten fold serial dilution of the water sample was prepared in sterile distilled water. The 10-2 dilution was used. From this, 1ml of sample was aseptically transferred to the centre of a prepared E.M.B agar. Using a sterile rod the water dropped was spread evenly on the media surface. The plates were incubated at 44.5°C for 24 hours. Lactose fermenting colonies formed were counted as faecal coliform in cfu/ml and the value multiplied by the dilution factor to get the actual level of the bacterial in each of the water samples collected.
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3.3.9
Faecal Streptococci Count
The multiple tube dilution technique as described by APHA (1998) using Azide dextrose broth was employed. One in ten dilution of the samples was made to 10-1. From this dilutions, 1ml was aseptically transferred to 9ml aliquots of sterile Azide dextrose broth and incubated at 37°C. It was examined for turbidity between 24 to 48 hours of incubation. Tube showing growth (turbid) was confirmed by streaking on Aesculin-azide agar and incubated at 37°C for 24 hours, brownish-black colonies with brown halo indicated the presence of faecal streptococci. This was confirmed by a negative reaction to catalase test. The number of colony forming units were counted and the values were multiplied by the dilution factor to calculate the actual microbial levels.
3.310 Preliminary Identification of the Isolates a. Isolation and Identification of selected Bacteria One milliliter (1 ml) of stock culture were mixed with 9.0 ml of peptone water as preenrichment and incubated at 37°C for 24 hours. The 24 hours culture was then streaked on to several selective media: MacConkey Agar, Salmonella-Shigella Agar, E.M.B Agar and Mannitol Salt Agar. b. Gram staining Colony of bacteria was smeared on slide and fixed. It was then smeared with crystal violet stain for 60 seconds. The stain was washed off with clean water. Lugol’s iodine was used to cover the smear for 60 seconds and washed off with clean water. This was followed by decolourization of the stain using alcohol and washed immediately with clean water. The smear was there after counter stained with neutral red stain for 2 minutes and washed off with 52
clean water. The smear was then placed in a draining rack to air-dry and examined microscopically with oil immersion objective to report the bacteria and cells. c. Growth on Selective Media The isolates were incubated for 24 hour at 37°C on the following selective media. The colour and morphology of the colonies were observed and noted. MacConkey agar was used to differentiate the lactose fermenters from the non-lactose fermenters, while Mannitol salt agar was used to presumptively identify S. aureus. Staphylococcus aureus appears as golden yellow colonies. On Eosin methylene blue agar, E. coli colonies appears greenish with metallic sheen. Salmonella-Shigella agar presumptively identify Salmonella spp. by the appearance of black centred colony. The isolates were further identified biochemically.
3.3.11 Biochemical Tests The following general biochemical tests were carried out according to the methods described by Cheesbrough (2006). a. Catalase test This test was carried out to differentiate between a catatase enzyme-producing bacterium such as Staphylococcus and non-catalase enzyme producing bacteria such as Streptococcus. Two (2 ml) of 3% hydrogen peroxide solution was measured and transferred into test tube. Using a sterile glass rod, several colonies of the test organisms were removed and immersed in the hydrogen peroxide solution. Immediate bubbling in the tube shows a positive catalase test.
53
b. Coagulase test This test was used to differentiate coagulase-producing Staphylococcus from the nonproducing ones. A drop of distilled water was placed on each end of a clean slide. Colonies of the test organisms were emulsified in each of the drops to make two thick suspensions. A loopful fresh human plasma was added to one of the suspension, and mixed gently. Clumping of the organisms within 10 minutes indicates a positive coagulase test. c. Oxidase test Oxidase test strip (Oxoid, England) was used. This test was used to differentiate between oxidase-positive and oxidase-negative bacteria. Several colonies of the test organisms were robbed on the strip using sterile glass rod. Formation of purple colouration within 5 seconds indicates a positive oxidase test.
3.3.12 Identification of the Oxidase-Negative Enterobacteriaceae Family a.
Microbact GNB 12E
The Microbact GNB 12E test strip is an identification system for Enterobacteriaceae that are oxidase-negative, nitrate-positive and glucose-fermenting Gram Negative Bacilli (GNB). It uses the ability of the isolates to ferment 12 sugars. The sugars included Inositol, Sorbitol, Rhamnose, Sucrose, Lactose, Arabinose, Adonitol, Raffinose, Glucose, Maltose, Dulcitol and Xylose. It contains 12 miniature biochemical tests. Organism identification is based on pH change and substrate utilization. The microplate is used for the identification of oxidasenegative, nitrate-positive glucose fermenter and is useful for screening pathogenic Enterobacteriaceae. It has 12 wells of individual substrate.
54
Using oxidase negative organism, 1 to 2 colonies were picked and emulsified in 3ml of sterile saline solution. This was mixed thoroughly to prepare homogenous suspension. The sealing tape was peeled backward and the strip placed in the holding tray. Sterile pipette was used to take 100µl of the bacteria suspension to fill each well. Wells 1, 2 and 3 were overlaid with 2 drops of sterile mineral oil. The inoculated row was there after resealed with the adhesive seal and appropriately labelled. This was done to the remaining rows with different organisms to be identified. The plate was then incubated at 37°C for 24 hours. At the end of the incubation period, two drops of Kovac’s reagent, one drop each of VP1 and VP11 and one drop of TDA were added to wells 8, 10 and 12 respectively before taking the readings. Colour changes of all the wells were compared with the standard colour chart provided by the manufacturer and number grade assigned to each well. Four digit coding of the sum of the positive results was then imputed into the Microbact Computer Aided Identification Package which interpreted the results by identifying the likely organisms with percentage share of the probability. b. Microbact Staphylococcal 12S This is based upon conventional identification system using a combination of sugar utilization and colorimetric enzyme detection substrate. Microbact Staphylocacal 12S can identify both coagulase- positive and coagulase-negative organisms. It is used in the identification of Gram-positive cocci, non-motile, non-spore forming, and catalase-positive facultative anaerobes. It has 12 tests. The organism identification is based on pH change and substrate utilization. Using the 24 hour pure culture of the organism to be identified, 2- 5 colonies were
55
picked and emulsified in 3 ml of Staphylococcus suspending medium. This was mixed thoroughly to prepare homogenous suspension. The lid from the test strip was removed and sterile micropipette was used to transfer 100µl of the bacterial suspension to fill each well. The well no 7 was overlaid with 2 drops of mineral oil. It was then incubated at 37°C for 24 hours. After the incubation 1 drop of Fast Blue reagent was added to well No.12 and the colour changes of all the wells were converted to numerical code which were then entered into Microbact Computer Aided Identification Package software for identification.
3.3.13
Preparation of Barium Sulphate Standard (McFarland 0.5)
One percent (v/v %) solution of sulphuric acid was prepared by adding 1 ml of concentrated sulphuric acid to 99 ml of water. One percent (w/v %) solution of barium chloride was then prepared by dissolving 0.5 g of dehydrated barium chloride (BaCl2.2H2O) in 50 ml of distilled water. A 0.6 ml of the barium chloride solution was added to 99.4 ml of the sulphuric acid solution and mixed. Small volume of the turbid solution was then transferred to a screw cap bottle of the same types as used for the test and control inocula (Cheesbrough, 2006).
3.3.14 Standardization of Innocula A sterile wire loop was used to pick discrete colonies of similar appearance from the overnight solid media culture and emulsified into 5 ml sterile physiological saline. The turbidity of the suspension was compared to the turbidity of the 0.5 McFarland standard and the suspension was adjusted by either adding more colonies of the bacteria or further diluting with sterile physiological saline to match the turbidity (Mahon et al., 1998). The suspended barium sulfate precipitate corresponds approximately to 1.5 × 108 cfu/ml (Madigan et al., 2006).
56
3.3.15 Antibiotic Susceptibility Testing The susceptibility of the different species of isolates was determined according to European Committee on Antimicrobial Susceptibility Testing, EUCAST (2014). A total of 10 antibiotics were used as test antibiotics, namely Chloramphenicol (30 µg), Gentamicin (10 µg), Ciprofloxacin (5 µg), Erythromycin (15 µg), Tetracycline (30 µg), Ampicillin (10 µg), Trimethoprim/Sulphamethoxazole (25 µg), Amoxicillin-clavulanate (30 µg), Cefuroxime sodium (30 µg) and Nitrofurantoin (300 µg). The standardized culture was streaked on to the surface of dried Mueller-Hinton agar in such a way as to ensure an even spread with sterile non-toxic cotton swab. The antibiotic discs under test were placed firmly on the surface of the agar using sterile forceps. The plates were allowed to stand for an hour to enable the antibiotics to diffuse into the agar. The plates were then incubated at 37°C for 18 hours. After the incubation, the plates were examined for the zones of inhibition, which were measured in millimeter using metric ruler. The result was interpreted using the interpretation criteria published by EUCAST (2014). The isolates were reported as sensitive (S), intermediate (I) and resistant (R) to the various antibiotics depending on the sizes of the zones of inhibition.
3.3.16 Determination of Multiple Antibiotic Resistance (MAR) Index The multiple antibiotic resistance (MAR) index was determined for each isolate as shown in the equation below. MAR index is the number of antibiotic(s) to which the organism is resistant divided by the total number of antibiotics tested (Krumperman, 1983; Paul et al., 1997). Number of Antibiotic(s) to which isolate was resistant MARI = Total number of antibiotics tested
57
3.3.17
Plasmid Curing
The method described by Udeze et al. (2012) was employed to cure the resistant isolates of plasmid they might contain. In this determination, the sub-Minimum Inhibitory Concentration (MIC) of acridine orange for each of the twenty-two multiple antibiotic resistant isolates was used. A 1.5g of acridine orange was weighed and dissolved in 150ml of sterile distilled water in a standard flask to have a concentration of 10mg/ml. Colonies from 24 hour cultures were emulsified in sterile normal saline to form a standard inoculum. Five millilitre, 5ml of double strength (DS) sterile nutrient broth was measured in the first test tube while the remaining nine test tubes had 5ml each of sterile single strength nutrient broth. Using a sterile micropipette 0.1ml of standard inoculum was added to all the test tubes. Five millilitre, 5ml of the stock acridine orange was measured and transferred into the first tube containing DS nutrient broth to have a concentration of 5000µg/ml. From the first tube, 5ml was transferred to the second tube and serially diluted up to the tenth tube which had concentration of 9.8µg/ml. The tubes were incubated at 37°C for 24 hours. Using the subMIC tube, the isolate was subcultured for 24 hours at 37°C. The susceptibility of the twenty two resistant isolates after acridine treatment was re-determined using agar diffusion method. The diameter of zones of inhibition was read and compared with the values before plasmid curing with acridine.
3.3.18 Molecular Characterization of some Antibiotic-Resistant Isolates The Molecular Diagnostic Laboratory of Veterinary Teaching Hospital, Ahmadu Bello University, Zaria, Nigeria was used in carrying out this experiment.
58
a. Bacterial Cell Preparation Nineteen (19) bacterial isolates after acridine treatment were used. Pure colonies of bacterial isolates were picked from a 24 hour culture and inoculated in 5 ml of sterile Luria-Bertani (LB) broth and incubated at 37°C for 24 hours. The bacterial cells were then harvested by centrifugation at 10,000 rpm for 5 minutes in an Eppendorff’s tube. The supernatant was discarded and cells harvested. The step was repeated for higher yield (Lephoto and Gray, 2013). b. Plasmid DNA Extraction The harvested cell pellets were vortexed in a Stuart vortex mixer for few seconds. i.
The harvested cells were re-suspended in 100µl of Buffer 1 (50mM Tris-HCl, 10mMEDTA, 100µg/mL RNas A, pH 8.0) and mixed properly to form homogenous suspension.
ii.
To each of the Eppendorff tube, 200µl of Buffer 2 (1% Sodium Dodecyl Sulphate,SDS, 0.2M NaOH) was added and the caps were closed. The solution was mixed and stored in freezer for 5 minutes.
iii.
A 150µl of ice-cold Buffer 3 (3.0M Potassium acetate, pH 5.2) was added to each Eppendorff tube, the caps were closed and then inverted for few times to mix the solution. The tubes were again kept on ice for 5 minutes after which they were placed in the centrifuge and spun at 10000rpm for 2 minutes. The supernatant was then transferred into clean 1.5ml Eppendorff tube and precipitate discarded.
iv.
To each tube of supernatant an equal volume (about 400µl) of isopropanol was added. The caps were closed and the content mixed vigorously. The tubes were left to stand 59
at room temperature for 2 minutes, and then centrifuged at 10000rpm for 5 minutes. The supernatant was carefully removed and discarded. v.
The DNA pellet was then washed with 200µl of 95% Ethanol and mixed by inversion several times. The tubes were again centrifuged at 10000rpm for 2-3 minutes. The supernatant was then carefully removed and discarded. The tubes were left with the caps open under vaccum for 15-20 minutes to dry off the last traces of Ethanol. c. Gel Electrophoresis
A 1.0g of agarose was weighed and dissolved in 100ml of TBA (Tris-Boric Acid) buffer and heated until agarose gel was fully dissolved. This make 1% Agarose gel. It was then cooled to about 45°C. Thereafter, the gel solution was poured into assembled gel holder with 20 place comb and allowed to solidify. The comb was wiggled a few times and lifted straight up. The gel holder lifted carefully and submerged in the electrophoresis tank containing TBA buffer with the wells closer to the negative electrode. Five microliter, 5.0µl each of loading dye was added to the plasmid preparations from the different bacterial isolates and loaded into different wells using micropipette. A standard 100bp molecular ladder was loaded into the first well. The gel was then run for 2 hours at 75Volts. The electrophoresis tank was disconnected and the gel containing separated plasmid was visualized under a Trans-illuminator UV light of wavelength 302nm. This was then photographed with a Polaroid camera and documented using BioRad gel electrophoresis documentation system. After photographing, the distance of migration of each isolate was determined relative to the standard DNA ladder loaded in the first well (Nworu et al., 2010).
60
The value of the DNA marker used was matched with the distance of migration of the bands in each isolates and the plasmid sizes of the isolates were determined using the Bio-rad image lab software.
3.3.19
Physicochemical Analysis of the Water Samples
Physicochemical properties of water were determined using standard methods described by American Public Health Association, APHA (1998). a. pH Determination The pH meter (Jenway 3505 bench top) was calibrated in accordance with manufacturer’s specification using standard solutions of pH 1,4,7,9 and 12. The electrode of the pH meter was rinsed with distilled water before and after each use. Twenty five millilitres (25ml) of each water samples was placed in a beaker and the electrode immersed. After stabilization, the pH value was read and recorded. For each sample, two readings was taken and the average calculated. b. Conductivity Measurement Prior calibration of meter (Jenway 4010, portable conductivity meter) was carried out with solution provided by manufacturer in accordance with instructions. Conductivity values (µS) of the water samples was measured using the conductivity meter. After priming, followed by washing with distilled water, the electrode was placed in a beaker containing 25ml of each samples in laboratory. Two sets of reading was obtained for each samples and average taken.
61
c. Temperature Measurement The temperature was taken at each point of sample collection using a mercury thermometer. Two reading were taken at each point before sample was collected and the average taken. d. Determination of Dissolved Oxygen (DO) A glass stoppered BOD bottle was used. At the site of sample collection, the BOD bottle was filled with water sample with no air trapped and stoppered. Two millilitre (2 ml) each of reagent A (Manganous sulphate solution) and reagent B (Alkaline potassium iodide solution) were added and stoppered. The BOD bottles were then transported to the Laboratory for immediate analysis. To the bottle, 2 ml sulphuric acid was added and shaken thoroughly. Ten millilitre (10 ml) of this solution was the transferred into a conical flask gently and a few drops of starch indicator was added. The solution was then titrated against 0.025N sodium thiosulphate solution and the end point noted when the initial blue colour turned colourless and the titre value read and recorded. DO was calculated thus: Tv × 0.025 × 8 × 1000 DO (mg/l) =
…………………..Eqn. 1 10
Where Tv is the titre volume used in titration e. Determination of Biochemical Oxygen Demand (BOD) Levels of biochemical oxygen demand (BOD) mg/l, which is the amount of oxygen required by bacteria while stabilizing decomposable organic matter under aerobic conditions, was measured by the difference in the dissolved oxygen content of sample on day one (D1) and 62
the dissolved oxygen content after five days of incubating the sample at 20 °C in the dark (D2). D1 - D2 BOD (mg/l) =
…………………….Eqn. 2 P
Where D1= dissolved oxygen of sample immediately after preparation. D2= dissolved oxygen of sample after 5 days of incubation P= decimal volumetric fraction of sample used. f. Determination of Chemical Oxygen Demand A 20ml of sample was measured into a flask of reflux unit and 10 ml of 0.025N potassium dichromate solution, a pinch of each silver nitrate and mercuric sulphate and 30 ml of sulphuric acid were added. The condenser was attached to flask and the flask heated on a heating mantle for two hours to reflux the contents. The flask was cooled and detached and its content diluted to 150ml by adding distilled water. A 2-3 drops of ferroin indicator solution was added and titrated against ferrous ammonium sulphate solution. At the end point, blue colour of content changed to reddish blue. Blank sample of distilled water was run simultaneously with this experiment. (Sample Tv – Blank Tv) × 0.025 × 8 × 100 COD (mg/l) =
...….Eqn. 3 Volume of sample used
Where Sample Tv = Titre volume of the sample Blank Tv = Titre volume of the blank
63
g. Determination of Total Alkalinity A 50ml of water sample was added into a conical flask followed by 2-3 drops of methyl orange indicator and titrated against 0.02N sulphuric acid until yellow colour solution turned orange (end point). Tv × 0.02 × 50,000 Toatal alkalinity (mg/l) =
…………… …..Eqn. 4 Volume of sample
Where Tv is the Titre volume h. Determination of Phosphate Content 100ml of sample was added into a volumetric flask followed by 1 drop of phenolphthalein indicator and 4ml of Vanadium molybdate reagent and then 10 drops of Stannous chloride reagent. The mixture was allowed to stand for 10 minutes and then measured photometrical at a wavelength of 690nm in a UV-Visible Spectrophotometer. From the calibration curve earlier produced using different concentrations (0.05, 0.15, 0.25, and 0.35 mg/l). The concentration of phosphate was computed using the formula: Weight of PO4 (mg) × 1000 ………….Eqn. 5
Phosphate (PO4 mg/l) = Volume of water sample (ml) i. Determination of Nitrate Content
A 25 ml of water sample was measured into porcelain basin and evaporated to dryness on a hot water bath. A 0.5 ml of phenol disulphuric acid was added to the residue. To the resulting solution, 5 ml of distilled water and 1.5 ml of potassium hydroxide solution was added and stirred for thorough mixing. The yellow colour supernatant was taken and the absorbance (S) 64
was read on a UV- visible spectrophotometer at 410nm wavelength. Distilled water was used as blank. Graph of absorbance against concentrations of various standard solutions was used to deduce the value of nitrate and expressed as mg/l.
3.3.20 Determination of Elemental Composition of Water and Fish Samples Digested water and fish samples were analysed using flame Atomic Absorption Spectrophotometer (model AA240FS, Varian) at Multi-user Laboratory, Department of Chemistry, Ahmadu Bello University Zaria, and the readings were recorded. The dilution factor of the sample was used to determine the final concentration of the various elements in water and fish samples.
65
CHAPTER FOUR 4.0
RESULTS
4.1
Bacteriological Analysis of River Lavun Water Samples
Figures 4.1 and 4.2 show the results of Heterotrophic Plate Count (HPC), Faecal Coliform Count (FCC) and Faecal Streptococci Count (FSC) of the water samples in April and September respectively. In April which is the beginning of raining season, HPC was 4.23, 4.38 and 4.48 at point A, B and C respectively. FSC at these points were lower than the HPC but more than FSC. HPC was higher at point C compared to FCC and FSC that were highest for the month at point A. However in September, these values are considerably higher. HPC was 5.64, 5.79 and 5.95 at point A, B and C respectively. A comparison of the HPC, FC and FS Counts at the different points showed that though there were slight increases or decreases, the differences were not really significant as shown in Figs. 4.1 and 4.2. The presentations showed that generally, the values of the three indices increased with increasing rainfall, with highest values in September as shown in Fig. 4.3. This trend was irrespective of the sampling points. The FC and FS values showed substantial increase by as much as 80% from the month of April to September of the same year.
4.2
Bacteriological Analysis of Fish
Heterotrophic Plate and Faecal Coliform Counts of fish caught from River Lavun are shown in Fig. 4.4. In April, HPC was 5.23 while FCC was far lower having value of 2.83.
66
4.38
4.23
4.50
4.48
5.00
4.00
3.00 2.43
2.56
2.62
2.50
HPC FCC
1.93
2.00
FSC 1.57
1.71
LOG. BACTERIA COUNT
3.50
1.50
1.00
0.50
0.00 POINT A
POINT B
POINT C
SAMPLING POINT
Fig. 4.1: The mean values of HPC, FCC and FSC in April, 2014 (Early rainy season). Point:
A = before the settlement B = beside the settlement C = after the settlement
Key:
HPC = Heterotrophic Plate Count FCC = Faecal Coliform Count FSC = Faecal Streptococci Count
67
5.79
5.64
6.00
5.95
7.00
2.53
2.40
3.00
3.83
3.85
HPC
2.76
4.00
3.66
LOG. BACTERIA COUNT
5.00
FCC FSC
2.00
1.00
0.00 POINT A
POINT B
POINT C
SAMPLING POINT
Fig. 4.2: The mean values of HPC, FCC and FSC in September, 2014 (Peak of rainy season). Point:
A = before the settlement B = beside the settlement C = after the settlement
Key:
HPC = Heterotrophic Plate Count FCC = Faecal Coliform Count FSC = Faecal Streptococci Count
68
5.95
7.00
3.83
4.00
2.53
April 3.00
1.57
2.43
LOG. BACTERIA COUNT
5.00
4.48
6.00
2.00
1.00
0.00 HPC
FCC
FSC
PARAMETERS (INDICATORS)
Fig. 4.3: Comparison of HPC, FCC and FSC at point C. Point:
A = before the settlement B = beside the settlement C = after the settlement
Key:
HPC = Heterotrophic Plate Count FCC = Faecal Coliform Count FSC = Faecal Streptococci count
69
September
5.23
5.45
5.20
5.18
5.23
6.00
5.73
7.00
2.58
HBC 2.73
2.81
2.63
3.00
2.67
4.00
2.83
LOG BACTERIAL COUNT
5.00
2.00
1.00
0.00 APRIL
MAY
JUNE
JULY
AUG.
SEP.
MONTH
Fig. 4.4: The mean values of HPC and FCC of fish from April to September, 2014. Key:
HPC = Heterotrophic Plate Count FCC = Faecal Coliform Count FSC = Faecal Streptococci Count
70
FCC
These values however decreased in May and June and increased again in July. The highest counts of HBC of 5.73 were obtained in September while FCC was highest in April. There was significant difference between the HBC and FCC of fish at p=0.011. However, when compared with the HBC and FCC of the water samples the difference was statistically not significant at p=0.801 and p=0.35 respectively.
4.3
Distribution of Isolates in Water and Fish Samples
Forty seven (47) isolates belonging to fifteen bacterial species were identified in the water samples. In the fish samples, on the other hand, 53 isolates belonging to 20 different species were identified. Eleven of the fifteen species identified in the water samples were also identified in the fish samples. Organisms identified from fish and water samples belong essentially to the same genera, mostly to Enterobacteriaceae family (91.5%) in water samples and 69.8% for fish samples). The other isolates were staphylococcus species. Of the Staphylococcus isolates only three different species were identified in the water samples as against eight different species in the fish samples (Table 4.1 and Fig. 4.7). About 76% proportion of water isolates were same, when compared with isolates from fish. The Enterobacteriaceae family, presented in Table 4.1 has the highest number of isolates from both water and fish with percentage occurrence of 80%. Among the Enterobacteriaceae members isolated, Klebsiella spp. were the highest with 38.75% occurrence followed by Enterobacter spp. (21.25%) while Yersinia spp. was the least isolated (1.25%). The Grampositive organisms isolated were different species of Staphylococcus. Staphylococcus spp., accounting for 20% occurrence. Of the total Enterobacteriaceae members isolated from water samples, 46%, 19%, 12% and 9% were Klebsiella spp., Enterobacter spp., E. coli and Serratia spp. respectively (Fig. 4.5) while in fish samples percentage occurrences was 30%, 71
24%, 19%, 14% and 8% for Klebsiella spp., Enterobacter spp., E. coli, Salmonella spp. and Serratia spp. respectively (Fig. 4.6).
4.4
Antibiotic Susceptibility Profiles of the Isolates
The results of the antibiotic susceptibility tests of the isolates are presented in Tables 4.2 and 4.3. The susceptibility profiles of the Enterobacteriaceae isolates show that majority of the organisms were susceptible to the inhibitory activities of gentamicin, nitrofurantoin, ciprofroxacin, chloramphenicol and co-trimoxazole. Except with erythromycin, Citrobacter spp. were susceptible to all the antibiotics tested. Resistance to the Tetracyclines, Erythromycin and penicillins (ampicillin and amoxicillin-clavulanate) was relatively high especially with Enterobacter, Salmonella and Serratia isolates, while moderate resistance was exhibited by the isolates against cefuroxime (a cephalosporin) and co-trimoxazole. Klebsiella spp from water samples were found to be highly resistant to Ampicillin, amoxicillin-clavulanate, tetracycline, erythromycin and cefuroxime but susceptible to inhibitory action of nitrofurantoin, gentamicin, ciprofloxacin, co-trimoxazole and chloramphenicol. In contrast, Klebsiella spp isolated from fish samples were susceptible to amoxicillin-clavulanate and cefuroxime but higher resistance to ampicillin, tetracycline and erythromycin when compared with that from water samples. The susceptibility profiles of the staphylococci isolates presented in Table 4.4 shows that the isolates were relatively resistant to ampicillin, amoxicillin-clavulanate, nitrofurantoin, gentamicin, ciprofloxacin, erythromycin, co-trimoxazole and chloramphenicol while
72
Table 4.1: Distribution of bacteria Isolates from water and fish samples collected from River Lavun Organism
Frequency
Total
Water
Fish
Enterobacter gergoviae
5
7
12
Enterobacter cloacae
2
0
2
Enterobacter sakazaki
0
1
1
Enterobacter aerogens
0
1
1
Enterobacter agglomerans
1
0
1
Serratia marcescens
2
1
3
Serratia rubidaea
0
2
2
Citrobacter freundii
3
1
4
Citrobacter diversus
1
0
1
Klebsiella pneumoniae
19
9
28
Klebsiella oxytoca
1
2
3
Escherichia coli
5
7
12
Salmonella sp
2
3
5
Salmonella arizonae
0
2
2
Shigella sonnei
1
1
2
Yersinia enterocolitica
1
0
1
Staphylococcus aureus
2
3
5
Staphylococcus xylosus
0
3
3
Staphylococcus saprophyticus
1
4
5
Staphylococcus simulans
1
2
3
Staphylococcus capitis subsp. Ureoly
0
1
1
Staphylococcus hominis
0
1
1
Staphylococcus auricularis
0
1
1
Staphylococcus chromogens
0
1
1
53
100
Total
47
73
Others 14%
Enterobacter spp. 19%
Escherichia coli 12%
Serratia spp. 9%
Klebsiella spp. 46%
Fig. 4.5: Composition of Enterobacteriaceae contaminants in water samples.
74
Others 5%
Salmonella spp. 14%
Enterobacter spp. 24%
Escherichia coli
Serratia spp.
19%
8%
Klebsiella spp. 30%
Fig. 4.6: Composition of Enterobacteriaceae contaminants in fish samples.
75
6
7
6
4
3
Fish
3
Water
2
3
2
FREQUENCY
4
5
2
0
0
1
0
S. X
S. A
S. S
OTHERS
ISOLATE
Fig. 4.7: Composition of Staphylococcus species contaminants in water and fish samples. S. X = Staphylococcus xylosus S. A = Staphylococcus aureus S. S = Staphylococcus saprophyticus
76
Table 4.2: Antibiotic Resistance Profiles of selected Enterobacteriacea Bacteria Isolated from River Lavun. Antibiotics
Percentage Resistant (%) Entr. sp
Citr. sp
Serr. sp
(n=8)
(n=4)
(n=2)
Ampicillin
75.00
Kleb. sp
E. coli
Salm. sp
(n=20)
(n=5)
(n=2)
25.00
100.00
90.00
60.00
50.00
100.00
65.00
0.00
50.00
Amox. /Clav.
75.00
25.00
Nitrofurantoin
0.00
0.00
0.00
5.00
0.00
0.00
Gentamicin
25.00
0.00
50.00
15.00
0.00
0.00
Ciprofloxacin
0.00
0.00
0.00
20.00
0.00
0.00
Tetracycline
75.00
25.00
100.00
75.00
80.00
50.00
Erythromycin
87.50
50.00
100.00
90.00
80.00
100.00
SMZ/TMP
0.00
25.00
0.00
30.00
60.00
50.00
Cefuroxime
50.00
25.00
100.00
60.00
0.00
50.00
25.00
0.00
5.00
20.00
50.00
Chloranphenicol 0.00
Amox. / Clav. : Amoxicillin / Clavulanic acid combination SMZ/TMP: Sulphamethoxazole trimethoprim combination Entr. = Enterobacter
Citr.= Citrobacter
Kleb. = Klebsiella
Salm. = Salmonella
77
Serr. = Serratia
Table 4.3: Antibiotic Resistance Profiles of selected Enterobacteriacea Bacteria Isolated from fish caught from River Lavun. Antibiotics
Percentage Resistant (%) Entr. sp
Serr. sp
(n=9)
(n=3)
Ampicillin
77.78
Amox. / Clav.
55.56
Kleb. sp
E. coli
(n=11)
(n=7)
(n=5)
100.00
71.43
80.00
33.33 66.67
Nitrofurantoin
0.00
0.00
Gentamicin
0.00
Ciprofloxacin
18.18
57.14
Salm. sp
80.00
0.00
28.57
0.00
66.66
18.18
28.57
0.00
0.00
0.00
9.09
0.00
0.00
Tetracycline
88.89
100.00
100.00
85.71
80.00
Erythromycin
00.00
100.00
100.00
85.71
100.00
SMZ/TMP
0.00
33.33
18.18
28.57
20.00
Cefuroxime
33.33
66.67
27.27
71.43
80.00
0.00
0.00
28.57
20.00
Chloramphenicol 11.11
Amox. / Clav. : Amoxicillin / Clavulanic acid combination SMZ/TMP: Sulphamethoxazole trimethoprim combination Entr. = Enterobacter
Serr. = Serratia
Kleb. = Klebsiella
Salm. = Salmonella
78
Table 4.4: Antibiotic Resistance Profiles of Staphylococcus spp. isolated from water and fish samples collected from River Lavun. Antibiotics
Percentage Resistant (%) Staph. xylosus (n=3)
Staph. aureus (n=5)
Staph. simolans (n=3)
Staph. saproph. (n=5)
Others (n=4)
Ampicillin
0.00
0.00
33.33
20.00
0.00
Amox. /Clav.
0.00
0.00
0.00
20.00
0.00
Nitrofurantoin
0.00
20.00
0.00
0.00
0.00
Gentamicin
33.33
60.00
33.33
20.00
25.00
Ciprofloxacin
0.00
0.00
0.00
0.00
25.00
Tetracycline
33.33
0.00
33.33
20.00
25.00
Erythromycin
0.00
0.00
0.00
0.00
0.00
SMZ/TMP
0.00
0.00
0.00
0.00
0.00
Cefuroxime
33.33
60.00
33.33
20.00
50.00
0.00
33.33
0.00
0.00
Chloramphenicol 0.00
Amox. / Clav. : Amoxicillin / Clavulanic acid combination. SMZ/TMP: Sulphamethoxazole trimethoprim combination. Staph. = Staphylococcus saproph. = saprophyticus
79
moderate resistance was exhibited against tetracycline and cefuroxime. Staphylococcus spp. irrespective of the source were generally susceptible to almost all the antibiotics tested.
4.5
Determination of MAR Index
MAR indices presented on Table 4.5 shows that most of the isolates are multi-drug resistant being resistant to two or more classes of the antibiotics tested. The highest MAR index of 0.7 were observed with Klebsiella and E. coli isolates. All the Serratia isolates were multiple drug resistant. Mojority of the Klebsiella isolates (87.1%), 83.4% of Enterobacter, 75.0% of E.coli and 60.0% of Citrobacter were found to be multi-drug resistant. In contrast, only a few of Staphylococci isolates (10.0%) were multiple drug resistant.
4.6
Resistance Pattern of Some Antibiotic Resistant Bacteria Species Isolated From River Water And Fish Caught From River Lavun.
Results of the plasmid curing of some isolates found to be multi-drug resistant is presented in Tables 4.6 and 4.7. As shown in these tables, the sensitivity of most of the isolates to some of the test antibiotics changed. A number of the isolates became sensitive to at least two (2) more drugs. Increased sensitivity was exhibited against Amoxicillin- clavulanate, Cefuroxime, Gentamicin, Nitrofurantoin, Chloramphenicol and Co-trimoxazole. Two Klepsiella pneumonae and an Escherichia coli did not show any change in number nor pattern of antibiotic resistance. After plasmid curing there were decreases in the number of isolates that were still resistant however these decreases were only 4.5% (each of Tetracyline and Erythromycin), 9.1% (Ampicillin) and 23.1% in Co-trimoxazole. Fourteen and ten more isolates became more sensitive to Cefuroxime and Amoxicillin respectively. 80
Table 4.5: MARI of bacteria isolates from water and fish samples collected from River Lavun. MARI
No. of organisms with MARI value Entr.
Serr.
Kleb.
Citr.
sp.
sp.
sp.
sp.
(n=17)
(n=5)
0.0
0
0
0.1
2
0.2
(n=31)
E. coli
Salm. sp.
Shig.
Staph.
sp.
sp.
(n=5)
(n=12)
(n=7)
(n=2)
(n=20)
1
2
1
0
0
7
0
1
0
1
0
0
4
1
0
2
0
1
2
0
7
0.3
6
0
9
0
1
0
1
2
0.4
7
3
8
3
2
3
0
0
0.5
0
0
0
0
0
0
0
0
0.6
1
2
5
0
5
2
1
0
0.7
0
0
5
0
1
0
0
0
MARI: Multiple Antibiotic Resistance Index Entr. = Enterobacter
Citr. = Citrobacter
Kleb. = Klebsiella
Salm. = Salmonella
Staph. = Staphylococcus
81
Serr. = Serratia Shig. = Shigella
Table 4.6: Resistance Pattern of Selected Antibiotic Resistant Bacteria Species Isolate From Water and Fish Samples Collected from River Lavun Before and After Curing. Isolate No
No of Antibiotics Resistant to Organism
Before curing
After curing
2
Serratia marcescens
6
3
Escherichia coli
6
3
7
Klebsiella pneumoniae
6
3
20
Klebsiella pneumoniae
7
4
21
Enterobacter gergoviae
6
4
24
Klebsiella pneumoniae
6
5
25
Klebsiella pneumoniae
5
4
43
Salmonella spp.
6
3
45
Escherichia coli
7
7
47
Escherichia coli
6
3
48
Escherichia coli
5
4
61
Serratia rubidaea
6
4
62
Escherichia coli
6
4
66
Klebsiella pneumoniae
7
4
68
Klebsiella pneumoniae
6
6
69
Klebsiella pneumoniae
5
4
70
Klebsiella pneumoniae
7
4
71
Klebsiella pneumoniae
7
7
73
Klebsiella pneumoniae
5
3
75
Salmonella spp.
6
3
76
Shigella sonnei
6
4
79
Escherichia coli
5
4
82
3
Table 4.7: Comparison of resistance of some antibiotics to twenty-two Multiple Antibiotic Resistant bacterial species isolated from water and fish caught from River Lavun before and after plasmid curing. Antibiotics
No. of Isolates Resistant out of 22 Before Plasmid curing
After Plasmid curing
% Decrease
Ampicillin
22/22
20/22
9.1
Amox. / Clav.
16/22
6/22
62.5
Nitrofurantoin
3/22
0/22
100
Gentamicin
7/22
2/22
71.4
Ciprofloxacin
4/22
2/22
50.0
Tetracycline
22/22
21/22
4.5
Erythromycine
22/22
21/22
4.5
SMZ/TMP
13/22
10/22
23.1
Cefuroxime
19/22
5/22
73.7
Chloramphenicol
6/22
3/22
50.0
Amox. / Clav. : Amoxicillin / Clavulanic acid combination. SMZ/TMP: Sulphamethoxazole trimethoprim combination.
83
4.7
Plasmid Analysis
Of the nineteen strains belonging to different species selected for the DNA plasmid analysis and subsequently electrophoresis, 68.4% (13 isolates) showed one or two plasmid bands and their molecular weight was in the range of 1637-3175bp (Table 4.8; Plate 4.1) while 31.6% (6 isolates) showed no visible bands. Of the total eight Klebsiella pneumoniae, 62.5% showed band weights ranging between 1.671 to 3.175kbp. Majority of the E. coli isolates (60%) had their band sizes to be above 2kbp.
4.8
Physicochemical Properties of Water Samples
Results of the physicochemical tests carried out on water samples from River Lavun at different points during the period of April, 2014 to September, 2014 are presented in Fig. 4.8, 4.9 and 4.10 and Appendices VIII to XII. The trend of DO, BOD and COD is shown on Fig. 4.8. DO ranges between 4mg/L and 16mg/L. The value decreased from April to May and started to rise in June to highest value of 16mg/L in September which is the peak of raining season. The BOD values showed similar pattern of the DO and it ranges from 4mg/L in April (Early raining season) to 13mg/L in September (Peak of raining season). COD values were generally high and as much as twice of the values of DO and BOD of the same month. The highest value of COD was 22.2mg/L and was obtained in July. The DO, BOD and COD during the sampling period showed similarity in the pattern of the values and the three parameters were generally lower during the months of April – June compared with values during the raining period (July – September). The increase in the values between these two periods were generally significant.
84
Plate 4.1: 1.0% agarose gel electrophoresis of 100 base pair and plasmid DNA isolated from Multidrug Resistant Isolates from River Water and fish caught from River Lavun. Key: Lane 1: Marker standard
Lane 11: Isolate 48. Escherichia coli
Lane 2: Isolate2. Serratia marcescens
Lane 12: Isolate 61. Serratia rubidaea
Lane 3: Isolate 3. Escherichia coli
Lane 13: Isolate 62. Escherichia coli
Lane 4: Isolate 7. Klebsiella pneumoniae
Lane 14: Isolate 66. Klebsiella pneumoniae
Lane 5: Isolate 20. Klebsiella pneumoniae
Lane 15: Isolate 69. Klebsiella pneumoniae
Lane 6: Isolate 21. Enterobacter gergoviae Lane 16: Isolate 70. Klebsiella pneumoniae Lane 7: Isolate 24. Klebsiella pneumonia
Lane 17: Isolate 73. Klebsiella pneumoniae
Lane 8: Isolate 25. Klebsiella pneumonia
Lane 18: Isolate 75. Salmonella spp
Lane 9: Isolate 43. Salmonella spp
Lane 19: Isolate 76. Shigella sonnei
Lane 10: Isolate 47. Escherichia coli
Lane 20: Isolate 79. Escherichia coli
85
Table 4.8: Comparison of Plasmid number and sizes of some antibiotic resistant bacteria isolated from water and fish caught from River Lavun. Isolate
Isolates
No
No of Plasmid Bands
Plasmid sizes (bp)
2
Serratia marcescens
1
2,613
3
Escherichia coli
2
1,637; 2,633
7
Klebsiella pneumoniae
2
1,671; 2,709
20
Klebsiella pneumoniae
1
2,746
21
Enterobacter gergoviae
1
2,784
24
Klebsiella pneumoniae
25
Klebsiella pneumoniae
0
43
Salmonella spp
2
1,754; 2,922
47
Escherichia coli
1
2,983
48
Escherichia coli
1
3,025
61
Serratia rubidaea
2
1,828; 3,067
62
Escherichia coli
0
Nil
66
Klebsiella pneumoniae
2
1,932; 3,175
69
Klebsiella pneumoniae
1
1,986
70
Klebsiella pneumoniae
0
Nil
73
Klebsiella pneumoniae
0
Nil
75
Salmonella spp
0
Nil
76
Shigella sonnei
1
2,142
79
Escherichia coli
0
Nil
1
86
1,778 Nil
pH, Alkalinity and Conductance values were presented in Fig. 4.9. pH values ranged between 6.63mg/L and 8.29mg/L. The slightly acidic values (6.63 - 6.79) were obtained during the peak of raining season while the values (8.09 – 8.29) obtained in the beginning of raining season were slightly basic. Alkalinity values ranged from 14mg/L (July) to 25mg/L (May). The Alkalinity values were generally higher than the pH values during this sampling period. The conductance value was highest (53µS/cm) in April in the beginning of raining season. There was sharp decrease in the values of conductance in July and this was approximately maintained through September. As shown in Fig. 4.8, conductivity values during the preraining period were significantly different and followed similar pattern with observation made with pH values. As shown in Fig. 4.10, while the nitrate content remained constant from April – September, the Phosphate content significantly increased by July up till September. The increment was also significant. Temperature during the sampling period ranged from 29°C during the peak of rainy season to 33°C in the early raining season. The values were inversely proportional to the values of DO observed. Statistical analyses using paired t- test show that there was no significant difference in pH (p=0.004) and Phosphate concentration (p=0.002) while the reverse was the case in DO (p=0.063), BOD (p=0.138), COD (p=0.185), Conductance (p=0.188), Alkalinity (p=0.122) and Nitrate (p=0.803) having higher values than the chosen α-value (0.05).
87
18.6
20.4
22.2
25
13
14
13.5
10
12
VALUE
15
13.2
14.4
16
20
DO 9
10
BOD
2
3
4
5
4
5
6
COD
0 APRIL
MAY
JUNE
JULY
AUG.
SEPT.
MONTH
Fig. 4.8: The mean values of DO, BOD and COD at River Lavun from April to September, 2014.
88
51
53
60
48
50
34
35
36
40
pH 30 25
Conductance (µS/cm)
20
20
22
Alkalinity (mg/L)
6.79
6.69
6.63
8.09
8.21
10
8.29
14
18
20
0 APRIL
MAY
JUNE
JULY
AUG.
SEPT.
Fig. 4.9: The mean values of pH, Conductance and Alkalinity at River Lavun from April to September, 2014.
89
2.26
2.23
2.3
2.5
1
1.1
1.5
1
Phosphate
0.9
VALUE (MG/L)
2
0.3
0.3
0.29
0.3
0.31
0.5
0.28
Nitrate
AUG.
SEPT.
0 APRIL
MAY
JUNE
JULY
MONTH
Fig. 4.10: The mean values of Nitrate and Phosphate at River Lavun from April to September, 2014.
90
4.9
Elemental Analysis of Water and Fish Samples
Composition of elements in the river water samples from different sampling points are presented in Tables 4.9 and Appendix XIV. This table show that Ag, Co and Pb were below detectable level during the sampling period irrespective of the sampling points. At point A, in August, except for Potassium, K, Sadium, Na and Cacium, Ca, concentration of other elements were relatively lower. Points B and C showed similar pattern of elemental concentration from April to August. However, as shown in Fig. 4.11, concentration of elements at point B was generally higher when compared with that of points A and C. Fig. 4.12 showed the concentration of some elements at point C from April to August. Except for K and Na which increased in concentration from April to August, concentrations of other elements decreased with increasing rainfall. Concentration of these elements in fish is presented in Table 4.10. In fish, Ag, Cd and Pb were below detectable limit. Co that was not detected in water had however accumulated in the fish. In most cases, concentration of elements in the sample fish decreases from April to August. Much higher values of Ca, K and Na compared with those of water samples are observed in the sample fish.
91
Table 4.9: Concentration of elements in water collected from River Lavun at sampling points B and C. Elements
Concentration (mg/L) April
B
June C
August
B
C 6.22
B
WHO Permissible Limit
C
Ca
6.68
7.86
6.17
Ag
0.00
0.00
0.00
0.00
0.00
0.00
-
Co
0.00
0.00
0.00
0.00
0.00
0.00
-
Fe
48.49 48.94
33.02 32.24
5.52
Cd
0.05
0.03
0.00
0.02
0.02
0.02
0.03
Mn
1.31 3.87
0.00
0.21
1.54
0.00
0.4
Pb
0.00 0.00
0.00 0.00
0.00
0.00
0.01
Zn
1.74 1.90
3.61 3.59
1.15
2.68
5.0
Ni
0.24 0.11
0.25
0.00
0.00
0.07
Mg
42.83 20.49
21.56 20.01
19.86 19.24
-
Cu
0.00 0.03
0.01 0.02
0.29
2.0
K
40.00 40.00
42.00 40.00
67.00 50.00
-
Na
53.00 52.00
53.00 51.00
98.00 74.00
200
0.10
22.78
A = Sampling point A B = Sampling point B
92
4.64
4.87
0.25
-
0.3
98
120
100
74 65
67
60
Point A
50
50
VALUE (MG/L)
80
Point B Point C
19.86 4.87
5.52
4
4.64
16
13.01
20
19.24
22.78
40
0 CALCIUM
IRON
MAGNESIUM
POTASIUM
SODIUM
ELEMENTS
Fig. 4.11: Concentrations of some elements in water samples at sampling points in August, 2014.
93
74
80
70
51
52
40
40
April
40 32.24
VALUES (MG/L)
50
50
48.94
60
June August
4.87
6.22
4.64
10
7.86
20
19.24
20.49
20.01
30
0 CALCIUM
IRON
MAGNESIUM
POTASIUM
SODIUM
ELEMENTS
Fig. 4.12: Concentration of some elements at sampling point C from April to August, 2014.
94
Table 4.10: Concentration of elements in fish collected from River Lavun at point C from April to August 2014. Elements
Concentration (mg/100g) April
Ca Ag
226.28±96.00 0.00
June
August
135.19±56.95 0.00
FAO/WHO Limit
36.86±11.85
-
0.00
-
Co
0.27±0.07
0.01±0.01
0.10±0.09
-
Fe
13.70±4.17
9.61±1.60
9.98±0.66
0.08
Cd
0.00
0.00
0.00
0.25
Mn
0.32±0.31
0.00
1.13±0.86
-
Pb
0.00
0.00
0.00
0.03
Zn
3.66±0.39
4.54±0.76
2.75±0.46
0.1
Ni
0.01±0.01
0.19±0.08
23.00±0.09
8.0
Mg
52.79±10.24
45.87±4.15
35.15±4.36
-
Cu
0.29±0.05
0.11±0.06
0.45±0.07
-
K
553.33±142.95
450.00±17.32
323.33±184.75
-
Na
230.00±20.00
163.33±15.28
130.00±20.00
-
95
CHAPTER FIVE 5.0
DISCUSSION
Rivers are natural sources of water which usually have their qualities altered through anthropogenic activities (such as farming, fishing, transportation and recreation) on or around the river, agricultural land runoffs, industrial effluents and municipal waste water discharges in to it. River Lavun is therefore not an exception and it is expected that the quality of River Lavun will be influenced by human activities, industrial and other environmental activities that take place along the river bank. The microbiological data obtained from this study clearly showed high heterotrophic, faecal coliform and faecal streptococci counts. The lower counts in the early part of the raining season was expected due to the fact that as rain fall becomes heavy and frequent, more pollutants (municipal, agricultural land runoff and stagnant ponds) find their way into the river through drainage and flooding. This trend has also been reported by Agbabiaka and Oyeyiola (2012). Similarly, Oyeleke and Istifanus (2008) observed that raining months recorded the highest counts of pollutants in the various sampling points on river Kaduna. Levels of faecal contaminants from human excreta followed similar trend. The heterotrophic and coliform levels obtained in this study are in agreement with the findings of Tytler (2011) and Olatunji et al. (2011). The relatively high counts of heterotrophic and faecal coliform obtained at point B might be due to additional contamination arising from washing, bathing, and swimming activities being carried out by the populace near this point. APHA (1998) reported that FC/FS ratio greater than 4.0 implies contamination arising mostly from human activity while values less than 1.5 indicate contamination from non-human sources such as domestic animals. The FC/FS ratio obtained in this study was between 4.88 96
and 28.00, an indication of human pollution. The populace around this river lack modern toilets and as such excrete on the land directly which are often washed into the river. These results are in agreement with those of Khalid et al. (2012) on Tigris River, Baghdad Province. Distribution of isolates in this study which showed that members of Enterobacteriaceae constituted 80% of the total bacteria and staphylococci, 20% is in agreement with that of Tytler (2011) who reported 62.44% of Enterobacteriaceae and 19.11% of Staphylococcus. This high prevalence of Enterobacteriaceae is also in line with the work of Yogendra et al. (2013) and Torimiro et al. (2014). Several researchers (Oyeleke and Istifanus, 2008; Raji and Ibrahim, 2011 and Novotny et al., 2004) had reported that most of the organisms isolated in this study are pathogenic and could cause water – food borne infections when ingested through contaminated water or food (fish). These bacteria have similarly been isolated from fish from Sokoto River (Salihu et al., 2012). Bacterial flora of fish depicts the levels of contamination of the water environment (Torimiro et al., 2014). Fish caught from this river have relatively highest heterotrophic and faecal counts during the peak of rainy season which might have resulted from heavy river contamination. However, microbial loads of fish were generally higher than that of water due to some level of additional contamination by the fishermen after the fish were caught probably due to unhygienic post-processing such as smoking and air drying. The type of microorganism found associated with fish depends on the aquatic habitat of fish and are known to be affected by certain factors like salinity level and bacterial load of the habitant (Diler et al., 2000). In fish the non-indigenous pathogens may not be pathogenic but could cause infection if ingested by man. Novotny et al. (2004) reported that food-borne 97
pathogens associated with fish and fish products include Clostridium butulinum type E and Vibrio parahaemolytiens. Other potentially human pathogenic bacteria associated with fish include C. perfringes, Staph. spp., Salmonella spp., Shigella spp., V. chholerae and other vibrios. Outbreaks usually occur due to the ingestion of insufficiently heat-treated fish or products contaminated after/during their processing (US DAFSIS, 2011). The higher pathogenic bacteria isolated from fish would have resulted from handling after fish were caught. This was supported by the isolation of fewer species of Staphylococcus from water (20%) as against 80% isolated from fish. Further contamination of fish by handling, and processing has been also reported by Novotny et al. (2004) as being a cause of food poisoning associated with fish. Salmonella spp from water samples exhibited resistant to 70% of the antibiotics tested followed by Serratia spp (60%) and 50% each by Enterobacter spp and Klebsiella spp. However, isolates from fish samples showed resistance to fewer number of antibiotics except with E. coli where it was resistant to 50% as against 40% of E. coli isolates from water samples. Awe and Ohikere (2014) reported higher resistant values in a study carried out on River Niger, North Central Nigeria, they revealed that Escherichia coli isolates were resistant to 80% of the test antibiotics. Most Enterobacteriaceae isolates from both water and fish are multiple antibiotic resistant, MAR. Unfortunately, 82.5% of the isolates had MAR index to be 0.3 and above, implying that the study area is associated as potential source of infectious outbreak. The highest MAR index of 0.7 was seen with the Klebsiella isolates from water, a worrying development as
98
Klebsiella isolates is a well-known pathogen. Effective management of infection that may arise from this organism is thus a problem. The present findings are in agreement with those reported by Tytler (2011) on River Kaduna, Northern Nigeria which reports that 58.6% of the Enterobacteriaceae isolates were MAR. Contaminated drinking water and food are major sources of enteric pathogens, causing several waterborne disease outbreaks. Consumption of the water with presence of antibioticresistant bacteria is a major public health concern as antibiotic-resistant bacteria could be transferred to humans, contributing to the spread and persistence of antibiotic-resistant bacteria in environments (Tao et al., 2010; Olayiwola and Adedokun, 2015). Presence of multiple antibiotic resistance among enteric bacteria isolates from aquatic environment has also been reported by Mervat et al. (2012), who investigated antimicrobial resistance profiles of Enterobateriaceae isolated from Rosetta Branch of River Nile, Egypt. Plasmid curing using acridine orange increased susceptibility to antibiotic from 62% to 100% depending on the antibiotic. This showed that a large proportion of the initial resistance was plasmid-mediated. Plasmid replication is inhibited by various agents that can intercalate between the bases of DNA, particularly acridine orange, without inhibiting the chromosomal DNA replication. Such inhibition can lead to loss of the plasmid (Freifelder, 1987). The results of physicochemical analysis of the River Lavun revealed that variation in climatic season have some effect on the physicochemical properties. Temperature is one of the most important ecological features and it controls behavioural characteristics of organisms, solubility of gases and salts in water. It helps in determination of other factors like pH,
99
conductivity, and various forms of alkalinity. The variation observed is mainly related with the temperature of atmosphere and weather conditions (APHA, 1998). In natural waters, CO2, H2CO3 and HCO3- are principal components that regulate pH (APHA, 1995). The lower pH values in the peak of rainy season, might be due to high organic content from the urban and agricultural runoff into the water body. Cude (2001) reported that water containing high organic content such as abattoir wastes tends to be acidic. Electrical conductivity reflects the overall degree of mineralization, and indicates the salinity of the water. Presence of ions, and their concentrations, mobility, and valence determines the electrical conductivity. The conductivity values are within the standards for drinking water (WHO, 2011). Nitrate levels were generally low, indicating that sources of nitrate pollution such as fertilizers and waste water discharges were not contributing factor in the study area. Alkalinity is the quantitative capacity of water sample to neutralize a strong acid to a designated pH (Gupta et al., 2003). In the present study, the lower alkalinity values in the peak period of rainy season may be due to increased (APHA, 1998). This result is in agreement with the work of Ashish and Yogendra (2009) who observed relatively lower alkalinity value (91mg/L) in the rainy season as against 200mg/L in winter. Dissolved oxygen is a factor which is used to determine whether the biological changes are brought about by aerobic or anaerobic organism. The slightly higher DO values in the peak of the rainy season indicate relatively mild organic pollution that might have resulted from sewage wastes from human settlement. (Igbal et al., 2006). These results also agrees with those of (Ashish and Yogendra, 2009; Yogendra et al., 2013).
100
Values of BOD which is a measure of oxygen in the water that is required by the aerobic organisms, shows the level of biodegradation of organic materials. The higher values in the peak of the rainy season also indicates presence of organic pollution. Chemical oxygen demand is commonly used to indirectly measure the amount of organic compounds in water (Kumar et al., 2011). It points to a deterioration of the water quality caused by the discharge of industrial effluent and domestic sewage (Gupta et al., 2003). The COD values in this study followed similar trend with BOD values. Moderate COD values indicate industrial pollution. The only industrial plants relatively near the study area are the Niger state water treatment plant and plastic industry situated in Minna, about one hundred kilometres away. During the period of this study, Ag, Co and Pb were not detected in the water samples analysed. Mn was only detected at points B and C. However, in the fish. Ag, Cd and Pb were below detectable limit while Co which was not found in water was detected in fish due to bio-accumulation. Iron (Fe) is one of the most abundant metals in the earth’s crust. It’s an essential trace element required by all forms of life. The average daily intake of iron has been estimated to be 17mg/day for males and 9-12mg/day for females (FAO/WHO, 2011). Hazard of pathogenic organisms may be increased because of the presence of Fe since most of these organisms need Fe for their growth (Tiwana et al., 2005). The Fe values of the different sites of river water samples and the fish were found to be above the permissible limit of standards for drinking water by (WHO, 2011) and Nigeria Industrial Standard, NIS (2007). These high Fe values observed might be due to the run-off from domestic and urban 101
wastes. This could lead to toxicity. Though excessive iron is not stored in the body, impaired ability to regulate iron absorption may result in siderosis in liver, pancreas, adrenals, thyroid, pituitary and heart and which could manifest as cirrhosis, adrenal insufficiency, heart failure or diabetics. The Mn levels in the river exceed the permissible limit of standard of drinking water prescribed by NIS (2007). The higher Mn concentration may be attributed to the addition of agricultural run-off, sewage and domestic wastes in the river. Osunkiyesi (2012) and Olagoke and Olantunji (2014) have also reported contamination by Mn of Ogun River, Abeokuta, South-west Nigeria and River Lanzun, Bida, Northern Nigeria respectively. Epidemiological studies have reported adverse neurological effect following extended exposure to high levels of Mn in drinking water could result (WHO, 2011). Zinc is a ubiquitous metal present in the environment. The values though within the acceptable limit recommended by WHO, for drinking water, exceeded those recommended by NIS (2007). The higher value at point B could be as a result of increased human activities such as washing in this site. River Lanzun was reported by Olagoke and Olatunji (2014) to be contaminated with Zinc having values higher than the WHO limit. The work of Adeniyi et al. (2012) is in line with this study having reported Zn concentration of 38.24mg/kg in fish which exceeded the FAO/WHO (2011) limit of 1mg/kg. The Copper (Cu) values of water samples at all the sampling points were above the permissible limits set by NIS (2007) and WHO (2011) for drinking water and FAO/WHO (2011) in fish. Cu is a necessary nutrient for human health. High doses of Cu results in anaemia, liver and kidney damage, and stomach and intestinal irritation (Edwards, 2010).
102
The higher Cu concentration might be due to the addition of agricultural run-off, sewage and domestic wastes in the river. The water concentration of Ni was above the permissible limit set by both the WHO and NIS while the values obtained in fish were within the limit by US DAFSIS (1993). Allergic contact dermatitis is the most prevalent effect of nickel in the general population (WHO, 2011). Nickel (Ni) is a moderately toxic element and consumption of water or food with high Ni content may cause serious problems in observation of many diseases (Schiavino, 2005). The result from this study show that Cd level in water samples were above permissible level but below detectable level in the fish samples. Cd is widely known to be a highly toxic nonessential heavy metal and it does not have a role in biological process in living organisms. Thus even at low concentration, Cd is harmful to living organisms (Tsui and Wang, 2004). The levels of Cd present in the river water may be due to industrial and agricultural operations around the catchment of the study area. Accumulation of Cd in the kidney can lead to renal dysfunction. Macro elements analysed (Na, K, Mg and Ca) in water samples were generally lower than those in the fish samples. The levels in the water samples are within permissible limits. High concentration of these macro elements in fish observed in this study concurs with results of Joanna et al. (2009). The concentrations of K, Na, Ca, and Mg in their study were higher than the values in this work. This present study showed that the most abundant macro element present in the fish samples was potassium. This is in line with the result of Adeniyi et al. (2012).
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Elemental composition of water diffuses into fish and could accumulated in it. In a study conducted by Tulay et al. (2014) it was reported that levels of trace elements such as Al, B, Ba, Cr, Cu, Fe, Mn, Ni and Zn in various fish species collected from Sakarya region, Turkey, were found to be below limit values provided by Turkish Food Codex (TFC), FAO and WHO. At variance to Tulay et al. (2014), concentrations of heavy metals ( Cd and Fe) in fish collected from Densu River, Accra, Ghana were found to be above the maximum limits by WHO as well as FAO standard but Pb, Hg and As were below detectable limit (Tiimub and Mercy, 2013). Different fish samples from Kaduna River in Nigeria have been analysed for toxic elemental contaminants such as Hg, Cd, V, Zn and Fe which were identified in appreciable amount in all the fish samples studied (Nwaedozie, 1998). Variation recorded in the concentration of different nutritional components in the fish examined could have been as a result of the ability of the fish to absorb and convert the essential nutrients from the diet or water bodies where they live (Adewoye et al., 2003). Generally, concentration of elements at point B were higher than those of point A and Point C. Point B is frequently being polluted by the inhabitants through their anthropogenic activities. Generally, in a study of water quality during rainy season of River Senegal in Mauritania by Abdoulaye, Elkory and Mohammed (2013) it was reported that the values of Iron, Manganese, Zinc and Lead were within the tolerable values while those of Aluminium and Copper were above the prescribed limit by WHO. Similarly, Umunnakwe, Akagha and Aharanwa (2013) revealed that River Aba in South-East Nigeria had concentrations of 104
Copper and Iron above the allowable limits especially at Nigeria breweries, however Calcium, Magnesium and Manganese were within the acceptable limits by WHO. Osunkiyesi (2012) also reported that Ogun River, South-West Nigeria, had concentrations of Manganese, Sodium, Potassium, Iron and Copper to be out of desirable levels prescribed by WHO. Mohammed and Musa (2012) revealed that River Lanzun, Bida, Northern centrals Nigeria is contaminated with some elements. The elements were Manganese, Copper and Iron having concentrations above WHO permissible limit. In contrast, similar study on the same river by Olagoke and Olatunji (2014) reported that many elements analysed had concentrations that are within WHO standard, however, at sites where major brass works are carried out, Iron, Copper, Manganese, Zinc, Lead and Chromium have concentrations higher than WHO standard.
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CHAPTER SIX 6.0
SUMMARY, CONCLUSION AND RECOMMENDATION
6.1
Summary
Anthropogenic activities on and around River Lavun altered the physicochemical properties of the river and bacteriologically polluted the water and its fish. Therefore the study was aimed at determining the bacteriological quality and physicochemical properties of the River with a view to addressing the health problems that may arise from the consumption of the water or fish. River Lavun is on Latt. 9°08'21"N and Long. 5°49'57"E. It is about 20km from Bida and serves as main source of water to the populace living around it. Samples of water were collected from three points on the river, and three sample fish (Clarias gariepinus) from April to September, 2014 and analysed using standard procedures. Bacterial load of the water and the fish were assessed. Bacteriological contaminants were identified using Microbact rapid identification kit from Oxoid UK. Susceptibility of the bacterial isolates to selected antibiotics and plasmid DNA analysis of MAR isolates were determined. Physicochemical parameters, such as DO, BOD and COD were assessed and the elemental composition was carried out using AAS. Microbial load of the fish revealed higher values of heterotrophic and faecal coliform counts when compared with counts of the water samples throughout the study period. Among the bacteria isolated, 80% were member of Enterobacteriaceae while 20% were Staphylococci spp. The antibiotic susceptibility testing revealed that, ciprofloxacin, chloramphenicol and nitrofurantoin were the most active antibiotics against the 106
Enterobacteriaceae isolates while amoxicillin-clavulanate, chloramphenicol, erythromycin and nitrofurantoin had best inhibitory actions against Staphylococci spp. Generally, isolates from water showed higher resistance than those from the fish.There was high prevalence of multiple antibiotic resistant Enterobacteriaceae. After plasmid curing, more isolates became sensitive to nitrofurantoin (100%), cefuroxime (73.3%), gentamicin (71.4%) and amoxicillin-clavulanate. (62.5%). The pH, conductivity, phosphate and nitrate values were within acceptable limit of WHO and/or NIS standards. BOD and COD were slightly high, indicating presence of organic and moderate industrial pollution. Fe, Mn, Zn, Cu, Ni and Cd were above the acceptable limit of drinking water by WHO and/or NSI. In fish Ni was within the limit while Fe, Zn and Cu were above the permissible limit prescribed by FAO/WHO.
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6.2
Conclusion
River Lavun is polluted with disease-causing bacteria and therefore not fit for domestic purposes such as drinking and cooking without adequate treatment. Fish from this river are also contaminated with pathogenic enteric bacteria. A high number of the isolates particularly the Enterobacteriaceae are multiple drug resistant with attendant health implication. Commonly used antibiotics such as ampicillin, tetracycline, and erythromycin are relatively ineffective others such as quinolones, chloramphenicol and nitrofurantoin are effective alternatives. The presence of heavy metals such as Fe, Mn, Zn, Cu, Ni and Cd makes the River water unsafe for domestic use and consumption. Chemical pollution of the river is mostly from human and agricultural activities, and much less from industries.
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6.3
Recommendation
Water from River Lavun should be pre-treated such as boiling before its domestic utilization.
Routine and regular quality monitoring of the river water should be initiated and sustained.
Public enlightenment campaigns should be intensified to educate the people on the dangers inherent in consuming water from the river without any form of treatment.
There is also need to educate the fishermen and consumers on the danger associated with improper handling of fresh fish and consumption of raw or improperly cooked fish.
Sewage should be properly treated before discharge into aquatic ecosystem.
The number of high number of MAR bacteria in River Lavun may have ecological and public health implications. Therefore the need for further studies especially the possible resistant plasmids and its molecular characterization and the possibility of returning the resistance genes to the human population through water usage and/or fish consumption.
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119
APPENDICES Appendix I: River Water Heterotrophic (Standard) Plate Count (cfu/ml) Month
Points A
B
C
April
1.7 x 104
2.4 X 104
3.0 X 104
May
5.0 X 104
8.0 X 104
9.0 X 104
June
7.0 X 104
9.6 X 104
9.8 X 104
July
2.6 X 104
3.0 X 105
3.5 X 105
August
1.6 X 105
4.6 X 105
5.2 X 105
September
4.4 X 105
6.1 X 105
8.9 X 105
Appendix II: River Water Faecal Coliform Count (cfu/ml) Month
Points A
B
C
April
4.2 x 102
3.6 X 102
2.7 X 102
May
2.4 X 102
5.6 X 102
7.1 X 102
June
5.2 X 102
4.6 X 103
7.8 X 102
July
2.5 X 103
4.7 X 103
3.0 X 103
August
3.3 X 103
5.0 X 103
4.8 X 103
September
4.6 X 103
7.0 X 103
6.8 X 103
Appendix III: River Water Faecal Streptococci Count Plate (cfu/ml) Month
Points A
B
C
April
86
51
37
May
89
77
79
June
93
74
86
July
170
150
130
August
210
160
190
September
570
250
340
120
Appendix IV: Heterotrophic plate count (HPC) and Faecal coliform count (FCC) of fish caught from River Lavun. Month
HPC (cfu/g)
FCC (cfu/g)
April
1.7±0.4 × 105
6.8±2.4 × 102
May
1.5±0.3 × 105
4.7±2.4 × 102
June
1.6±0.3 × 105
4.3±1.8 × 102
July
2.8±0.6 × 105
6.4±2.0 × 102
August
1.7±0.3 × 105
5.4±1.1 × 102
September
5.4±0.6 × 105
3.8±1.3 × 102
Appendix V: EUCAST Break point, 2014 (Enterobacteriaceae) Antibiotic
S≥
I
R>
Ampicillin
14
0
14
Amoxiclav.
19
0
19
Cefuroxime
18
0
18
Ciprofloxacin
22
19-21
19
Gentamicin
17
14-16
14
Chloramphenicol
17
0
17
Nitrofurantoin
11
0
11
SXT
16
13-15
13
Tetracycline*
22
19-21
19
Erythromycin*
21
18-20
19
Note: S = Sensitive
I = Intermediate R = Resistant
*= No values for Enterobacteriaceae so values for Staphylococcus spp were used
121
Appendix VI: EUCAST Break points 2014 (Staphylococcus spp.) Antibiotic
S≥
I
R>
Ampicillin
18
0
18
Amoxiclav.
18
0
18
Cefuroxime
22
0
22
Ciprofloxacin
20
0
20
Gentamicin
22
0
22
Erythromycin
21
18-20
18
Tetracycline
22
19-21
19
Chloramphenicol
18
0
18
Nitrofurantoin
13
14-16
13
SXT
17
14-16
14
Note: S = Sensitive
I = Intermediate R = Resistant
122
Appendix VI: Interpretation of Antibiotic Susceptibility Tests of the different Bacterial Isolates. S/N
Isolate No
AMP AMC
F
CN
CIP
TE
E
SXT
CXM
C
1
1
R
R
S
S
S
R
R
S
R
S
2
2
R
R
S
R
S
R
R
S
R
S
3
3
R
R
R
R
S
R
R
S
S
S
4
4
R
R
S
R
I
R
R
S
S
S
5
5
R
R
S
S
S
R
R
S
R
S
6
6
S
S
S
S
S
I
I
S
S
S
7
7
R
R
S
R
S
R
R
S
R
S
8
8
S
S
S
S
S
S
R
S
S
S
9
9
R
R
S
S
S
R
R
S
S
S
10
10
R
R
S
I
S
R
R
S
S
S
11
11
R
R
S
S
S
R
R
S
S
S
12
12
R
R
S
S
S
R
R
S
R
S
13
13
S
S
S
S
S
S
S
S
S
S
14
14
R
R
S
R
S
R
R
S
S
S
15
15
S
R
S
R
S
R
R
S
S
S
16
16
R
R
S
S
S
R
R
S
R
S
17
17
R
R
S
I
S
R
R
S
S
S
18
18
S
S
S
I
S
I
R
S
S
S
19
19
R
S
S
I
S
R
R
S
S
S
20
20
R
R
R
S
S
R
R
S
R
R
21
21
R
R
S
R
S
R
R
S
R
S
22
22
R
R
S
S
S
I
R
S
S
S
23
23
R
R
S
S
S
R
R
S
S
S
24
24
R
R
S
R
S
R
R
S
R
S
25
25
R
S
S
R
S
R
R
S
R
S
26
26
R
S
S
R
S
I
R
S
R
S
27
27
R
S
S
S
S
R
R
S
S
S
28
28
S
R
S
S
S
I
R
S
S
S
29
29
S
R
S
I
S
S
R
S
R
R
123
30
30
R
S
S
S
R
R
R
S
S
S
31
31
R
S
S
S
S
R
R
S
S
S
32
32
R
S
S
I
S
R
R
S
S
S
33
33
S
R
S
S
S
R
R
I
S
R
34
34
R
R
S
S
S
S
R
S
S
S
35
35
S
S
S
S
S
R
R
S
R
R
36
36
R
R
S
S
S
R
R
S
S
S
37
37
R
S
S
S
S
R
R
S
S
S
38
38
R
S
S
S
S
R
R
S
S
S
39
39
R
S
S
S
S
R
R
R
S
S
40
40
S
S
S
R
S
R
R
S
R
S
41
41
R
S
S
S
S
R
R
R
S
S
42
42
R
R
S
I
S
R
R
S
R
S
43
43
R
R
S
S
S
R
R
R
R
S
44
44
R
R
S
S
S
R
R
S
R
S
45
45
R
R
S
S
S
R
R
R
R
R
46
46
R
R
S
S
S
R
R
S
R
S
47
47
R
R
S
R
S
R
R
S
R
S
48
48
R
S
S
S
S
R
R
R
R
S
49
49
R
R
S
S
S
R
R
S
R
S
50
50
R
S
S
S
S
R
R
S
R
S
51
51
R
S
S
S
S
R
R
S
S
S
52
52
S
S
S
S
S
R
R
S
S
S
53
53
R
S
S
I
S
R
R
S
S
S
54
54
R
R
S
S
S
R
R
S
R
S
55
55
R
S
S
S
S
R
R
S
R
S
56
56
R
R
S
S
S
R
R
S
R
S
57
57
R
R
S
S
S
R
R
S
S
S
58
58
R
R
S
S
S
I
R
S
S
S
59
59
S
S
S
S
I
I
S
S
R
S
60
60
R
S
S
S
S
R
R
R
S
S
124
61
61
R
R
S
S
S
R
R
R
R
S
62
62
R
S
R
S
S
R
S
S
R
R
63
63
S
S
S
S
S
S
S
S
S
S
64
64
S
S
S
S
S
S
S
S
S
S
65
65
S
S
S
S
S
R
S
S
S
S
66
66
R
R
S
I
R
R
R
R
R
S
67
67
R
R
S
I
I
R
R
S
R
S
68
68
R
S
S
S
R
R
R
R
R
S
69
69
R
S
S
S
S
R
R
R
R
S
70
70
R
R
S
S
R
R
R
R
R
S
71
71
R
R
S
I
R
R
R
R
R
S
72
72
R
S
S
S
S
R
R
R
S
S
73
73
R
S
S
I
S
R
R
R
R
S
74
74
R
S
S
S
S
I
R
S
R
S
75
75
R
R
S
S
S
R
R
R
R
R
76
76
R
R
S
S
I
R
R
R
S
R
77
77
S
S
S
I
S
S
S
S
S
S
78
78
R
S
S
S
S
I
R
S
S
S
79
79
R
S
S
S
S
R
R
R
S
R
80
80
R
S
S
S
S
R
R
R
S
S
81
81
S
S
S
S
S
S
S
S
R
R
82
82
S
S
S
S
S
I
S
S
S
S
83
83
R
S
S
R
S
S
I
S
R
S
84
84
S
S
R
R
S
S
S
S
S
S
85
85
S
S
S
S
S
I
S
S
R
S
86
86
S
S
S
R
S
S
S
S
R
S
87
87
S
S
S
S
S
S
S
S
S
S
88
88
S
S
S
S
S
S
S
S
S
S
89
89
S
S
S
R
S
R
S
S
S
S
90
90
S
S
S
S
S
S
S
S
S
S
91
91
R
R
S
S
S
S
S
S
R
S
125
92
92
S
S
S
R
S
R
S
S
R
S
93
93
S
S
S
S
S
S
S
S
S
S
94
94
S
S
S
S
S
S
S
S
S
S
95
95
S
S
S
S
S
I
S
S
S
S
96
96
S
S
S
S
S
R
S
S
S
R
97
97
S
S
S
S
R
S
S
S
S
S
98
98
S
S
S
R
S
I
S
S
R
S
99
99
S
S
S
S
S
I
S
S
R
S
100
100
S
S
S
S
S
I
S
S
R
S
NOTE: S=Sensitive I=Intermediate R=Resistants
126
Appendix VII: Resistant pattern of some Antibiotic Resistant bacteria species isolated from river water and fish caught from River Lavun. Isolate Isolates
Resistance Profile before curing
Resistance Profile before curing
No 2
Serratia spp
AMP-AMC-CN-TE-E-CXM
AMP-TE-E
3
Escherichia coli AMP-AMC-F-CN-TE-E
AMP-TE-E
7
Klebsiella spp
AMP-AMC-CN-TE-E-CXM
AMP-TE-E
20
Klebsiella spp
AMP-AMC-CN-TE-E-CXM-C
AMP-AMC-E-CXM
21
Entr. gergoviae
AMP-AMC-CN-TE-E-CXM
AMP-AMC-TE-E
24
Klebsiella spp
AMP-AMC-CN-TE-E-CXM
AMP-AMC-TE-E-CXM
25
Klebsiella spp
AMP-CN-TE-E-CXM
AMP-CN-TE-E
43
Salmonella spp
AMP-AMC-TE-E-SXT-CXM
AMP-TE-E
45
Escherichia coli AMP-AMC-TE-E-SXT-CXM-C AMP-AMC-TE-E-SXT-CXM-C
47
Escherichia coli
AMP-AMC-CN-TE-E-CXM
CN-TE-E
48
Escherichia coli
AMP-TE-E-SXT-CXM
AMP-TE-E-SXT
61
Serratia spp
AMP-AMC-TE-E-SXT-CXM
AMP-TE-E-SXT
62
Escherichia coli
AMP-F-TE-E-CXM-C
AMP-TE-E-C
66
Klebsiella spp
AMP-AMC-CIP-TE-E-SXT-CXM
AMP-TE-E-SXT
68
Klebsiella spp
AMP-CIP-TE-E-SXT-CXM
69
Klebsiella spp
AMP-TE-E-SXT-CXM
AMP-TE-E-SXT
70
Klebsiella spp
AMP-AMC-CIP-TE-E-SXT-CXM
AMP-TE-E-SXT
71
Klebs. Spp
73
Klebsiella spp
75
Salmonella spp
76
Shigella spp
AMP-AMC-TE-E-SXT-C
AMP-AMC-TE-C
79
Escherichia coli
AMP-TE-E-SXT-C
AMP-TE-E-SXT
AMP-CIP-TE-E-SXT-CXM
AMP-AMC-CIP-TE-E-SXT-CXM AMP-AMC-CIP-TE-E-SXT-CXM AMP-TE-E-SXT-CXM AMP-AMC-TE-E-SXT-CXM-C
127
AMP-TE-E TE-E-SXT
Appendix VIII: Physicochemical Parameters of April, 2014 Parameter (Unit) Ph Temperature (°C) Conductance (µS/cm) D.O (mg/L) B.O.D (mg/L) C.O.D (mg/L) Alkalinlty (mg/L) Phosphate (mg/L) Nitrate (mg/L)
A
B
C
7.85 32 44 8 6 16.8 20 0.9 0.32
8.19 33 48 6 4 14.3 22 0.85 0.19
8.29 33 53 6 4 14.4 20 0.9 0.28
NIS 6.5 - 8.5
1000
50
Appendix IX: Physicochemical Parameters of May, 2014 Parameter (Unit) Ph Temperature (°C) Conductance (µS/cm) D.O (mg/L) B.O.D (mg/L) C.O.D (mg/L) Alkalinlty (mg/L) Phosphate (mg/L) Nitrate (mg/L)
A
B
C
7.98 32 45 8 5 16 22 1.22 0.22
8.16 33 46 7 5 14.7 18 1.1 0.22
8.21 32 48 4 2 13.2 25 1 0.31
NIS 6.5 - 8.5
1000
50
Appendix X: Physicochemical Parameters of June, 2014 Parameter (Unit) Ph Temperature (°C) Conductance (µS/cm) D.O (mg/L) B.O.D (mg/L) C.O.D (mg/L) Alkalinlty (mg/L) Phosphate (mg/L) Nitrate (mg/L)
A
B
C
8.1 31 38 6 4 14.5 16 0.95 0.16
8.21 32 50 5 3 14.3 20 1.07 0.3
8.09 32 51 5 3 13.5 20 1.1 0.3
128
NIS 6.5 - 8.5
1000
50
Appendix XI: Physicochemical Parameters of July, 2014 Parameter (Unit) pH Temperature (°C) Conductance (µS/cm) D.O (mg/L) B.O.D (mg/L) C.O.D (mg/L) Alkalinlty (mg/L) Phosphate (mg/L) Nitrate (mg/L)
A
B
C
6.34 30 36 8 5 18 20 2.32 0.24
6.61 30 35 12 9 22 18 2.26 0.29
6.63 30 34 14 10 22.2 14 2.3 0.29
NIS 6.5 - 8.5
1000
50
Appendix XII: Physicochemical Parameters of August, 2014 Parameter (Unit) Ph Temperature (°C) Conductance (µS/cm) D.O (mg/L) B.O.D (mg/L) C.O.D (mg/L) Alkalinlty (mg/L) Phosphate (mg/L) Nitrate (mg/L)
A
B
C
6.62 29 35 6 3 15.5 16 2.24 0.3
6.65 29 86 10 6 23 16 2.09 0.31
6.69 30 36 12 9 20.4 22 2.23 0.3
NIS 6.5 - 8.5
1000
50
Appendix XII: Physico-chemical Parameters of September, 2014 Parameter (Unit) pH Temperature (°C) Conductance (µS/cm) D.O (mg/L) B.O.D (mg/L) C.O.D (mg/L) Alkalinlty (mg/L) Phosphate (mg/L) Nitrate (mg/L)
A
B
C
NIS
6.61 30 36 14 10 16.9 14 2.23 0.24
6.67 30 35 10 6 20 12 2.28 0.22
6.79 30 35 16 13 18.6 18 2.26 0.3
6.5 - 8.5
129
1000
50
Appendix XIII: Guidelines on drinking water and fish Element
*Water (mg/L)
**Water (mg/L)
***Fish (mg/100g)
Ca
-
-
-
Ag
-
-
-
Co
-
-
-
Fe
0.3
0.3
0.08
Cd
0.003
0.003
0.25
Mn
0.4
0.2
-
Pb
0.01
0.01
0.03
Zn
5.0
3.0
0.1
Ni
0.07
0.02
8.0
Mg
-
0.20
-
Cu
2.0
1.0
-
K
-
-
-
Na
200
200
-
Note:
* = WHO, 2011 ** = NIS, 2007 *** = FAO/WHO, 2011
130
Appendix XIV: Concentration of elements in water collected from River Lavun at
sampling Point A. Elements
Concentration (mg/L) April
June
August
Ca
2.23
4.81
13.01
-
Ag
0.00
0.00
0.00
-
Co
0.00
0.00
0.00
-
Fe
29.18
33.04
4.0
0.3
Cd
0.00
0.05
0.03
0.003
Mn
0.00
0.00
0.00
0.4
Pb
0.00
0.00
0.00
0.01
Zn
1.67
1.42
1.38
5.0
Ni
0.13
0.16
0.00
0.07
Mg
19.73
16.56
16.00
-
Cu
0.02
0.03
0.20
2.0
K
37.00
32.00
50.00
-
Na
49.00
43.00
65.00
200
131
WHO permissible limit
